Methods and devices for obtaining and assaying mammary fluid samples for evaluating breast diseases, including cancer

ABSTRACT

Non-invasive methods are provided for obtaining biological samples of mammary fluid or mammary fluid components by administering oxytocin to a patient to stimulate expression of mammary fluid. During or after mammary fluid expression, a biological sample is collected in the form of whole mammary fluid, whole cells or cellular components, other selected liquid or solid fractions of the mammary fluid, purified or bulk proteins, glycoproteins, peptides, nucleotides or other desired Constituents of mammary fluid. Methods and kits are also provided for determining the presence or amount of a breast disease marker in biological samples of mammary fluid or mammary fluid components obtained according to the above sample collection methods. Also provided within the invention are novel breast pump and breast pump adapter devices which incorporate a solid phase sample collection medium integrated within the breast pump or adapter or otherwise fluidly connected therewith. These devices collect a sample of expressed breast fluid by contacting the fluid with a solid phase sample collection medium while the pump or adapter is applied to the breast.

RELATED APPLICATIONS

This application is a continuation-in-part of and claims the benefit ofU.S. patent application Ser. No. 08/709,207 filed Aug. 27, 1996, nowU.S. Pat. No. 5,798,266, issued Aug. 25, 1998.

TECHNICAL FIELD

The invention relates to methods, devices, and kits for obtaining andassaying biological samples from mammary fluid. More specifically, theinvention relates to methods, devices, and kits for obtaining andassaying fluid and cytological samples from the mammary glands of amammalian subject for evaluating, diagnosing and managing breastdisease, including infections, pre-cancerous conditions, cancersusceptibility and cancer.

BACKGROUND OF THE INVENTION

Breast cancer is by far the most common form of cancer in women, and isthe second leading cause of cancer death in humans. Despite many recentadvances in diagnosing and treating breast cancer, the prevalence ofthis disease has been steadily rising at a rate of about 1 k per yearsince 1940. Today, the likelihood that a women living in North Americawill develop breast cancer during her lifetime is one in eight.

The current widespread use of mammography has resulted in improveddetection of breast cancer. Nonetheless, the death rate due to breastcancer has remained unchanged at about 27 deaths per 100,000 women. Alltoo often, breast cancer is discovered at a stage that is too faradvanced, when therapeutic options and survival rates are severelylimited. Accordingly, more sensitive and reliable methods are needed todetect small (less than 2 cm diameter), early stage, in situ carcinomasof the breast. Such methods should significantly improve breast cancersurvival, as suggested by the successful employment of Papinicolousmears for early detection and treatment of cervical cancer.

In addition to the problem of early detection, there remain seriousproblems in distinguishing between malignant and benign breast disease,in staging known breast cancers, and in differentiating betweendifferent types of breast cancers (eg. estrogen dependent versusnon-estrogen dependent tumors). Recent efforts to develop improvedmethods for breast cancer detection, staging and classification havefocused on a promising array of so-called cancer “markers.” Cancermarkers are typically proteins that are uniquely expressed (eg. as acell surface or secreted protein) by cancerous cells, or are expressedat measurably increased or decreased levels by cancerous cells comparedto normal cells. Other cancer markers can include specific DNA or RNAsequences marking deleterious genetic changes or alterations in thepatterns or levels of gene expression associated with particular formsof cancer.

A large number and variety of breast cancer markers have been identifiedto date, and many of these have been shown to have important value fordetermining prognostic and/or treatment-related variables. Prognosticvariables are those variables that serve to predict disease outcome,such as the likelihood or timing of relapse or survival.Treatment-related variables predict the likelihood of success or failureof a given therapeutic plan. Certain breast cancer markers clearly serveboth functions. For example, estrogen receptor levels are predictive ofrelapse and survival for breast cancer patients, independent oftreatment, and are also predictive of responsiveness to endocrinetherapy. Pertschuk et al., Cancer 66: 1663-1670, 1990; Parl and Posey,Hum. Pathol. 19: 960-966, 1988; Kinsel et al., Cancer Res. 49:1052-1056, 1989; Anderson and Poulson Cancer 65: 1901-1908, 1989.

The utility of specific breast cancer markers for screening anddiagnosis, staging and classification, monitoring and/or therapypurposes depends on the nature and activity of the marker in question.For general reviews of breast cancer markers, see Porter-Jordan et al.,Hematol. Oncol. Clin. North Amer. 8: 73-100, 1994; and Greiner,Pharmaceutical Tech., May, 1993, pp. 28-44. As reflected in thesereviews, a primary focus for developing breast cancer markers hascentered on the overlapping areas of tumorigenesis, tumor growth andcancer invasion. Tumorigenesis and tumor growth can be assessed using avariety of cell proliferation markers (for example Ki67, cyclin D1 andproliferating cell nuclear antigen (PCNA)), some of which may beimportant oncogenes as well. Tumor growth can also be evaluated using avariety of growth factor and hormone markers (for example estrogen,epidermal growth factor (EGF), erbB-2, transforming growth factor (TGF),which may be overexpressed, underexpressed or exhibit altered activityin cancer cells. By the same token, receptors of autocrine or exocrinegrowth factors and hormones (for example insulin growth factor (IGF)receptors, and EGF receptor) may also exhibit changes in expression oractivity associated with tumor growth. Lastly, tumor growth is supportedby angiogenesis involving the elaboration and growth of new bloodvessels and the concomitant expression of angiogenic factors that canserve as markers for tumorigenesis and tumor growth.

In addition to tumorigenic, proliferation and growth markers, a numberof markers have been identified that can serve as indicators ofinvasiveness and/or metastatic potential in a population of cancercells. These markers generally reflect altered interactions betweencancer cells and their surrounding microenvironment. For example, whencancer cells invade or metastasize, detectable changes may occur in theexpression or activity of cell adhesion or motility factors, examples ofwhich include the cancer markers Cathepsin D, plasminogen activators,collagenases and other factors. In addition, decreased expression oroverexpression of several putative tumor “suppressor” genes (for examplenm23, p53 and rb) has been directly associated with increased metastaticpotential or deregulation of growth predictive of poor disease outcome.

Additional representative breast disease markers within these variousclasses include prostaglandin E2 (PGE2); estrogen-regulated proteinssuch as pS2; interleukins (eg., IL-10); S-100 protein; vimentin;epithelial membrane antigen; prostate specific antigen (PSA); bcl-2;CA15-3 (an aberrant form of polymorphic epithelial mucin (PEM)); CA19-9; mucin core carbohydrates (eg., Tn antigen and Tn-like antigens);alpha-lactalbumin; lipid-associated sialic acid (LASA);galactose-N-acetylgalactosamine (Gal-GalNAC); GCDFP-15; Le(y)-relatedcarbohydrate antigen; CA 125; urokinase-type plasminogen activator (uPA)and uPA related antigens and complexes (eg., LMW-uPA, HMW-uPA, uPAaminoterminal fragment (ATF), uPA receptor (uPAR) and complexes withinhibitors such as PAl-1 and PAl-2); beta-glucuronidase; CD31; CD44splice variants; blood group antigens (eg., ABH, Lewis, and MN); and

genetic lesions or altered expression levels of CCND1, EMS1, BRCA1 andBRCA2 genes.

In summary, the evaluation of proliferation markers, oncogenes, growthfactors and growth factor receptors, angiogenic factors, proteases,adhesion factors and tumor suppressor genes, among other cancer markers,can provide important information concerning the risk, presence, statusor future behavior of cancer in a patient. Determining the presence orlevel of expression or activity of one or more of these cancer markerscan aid in the differential diagnosis of patients with uncertainclinical abnormalities, for example by distinguishing malignant frombenign abnormalities.

Furthermore, in patients presenting with established malignancy, cancermarkers can be useful to predict the risk of future relapse, or thelikelihood of response in a particular patient to a selected therapeuticcourse. Even more specific information can be obtained by analyzinghighly specific cancer markers, or combinations of markers, which maypredict responsiveness of a patient to specific drugs or treatmentoptions.

Methods for detecting and measuring cancer markers have been recentlyrevolutionized by the development of immunological assays, particularlyby assays that utilize monoclonal antibody technology. Previously, manycancer markers could only be detected or measured using conventionalbiochemical assay methods, which generally require large test samplesand are therefore unsuitable in most clinical applications. In contrast,modern immunoassay techniques can detect and measure cancer markers inrelatively much smaller samples, particularly when monoclonal antibodiesthat specifically recognize a targeted marker protein are used.Accordingly, it is now routine to assay for the presence or absence,level, or activity of selected cancer markers by immunohistochemicallystaining breast tissue specimens obtained via conventional biopsymethods. Because of the highly sensitive nature of immunohistochemicalstaining, these methods have also been successfully employed to detectand measure cancer markers in smaller, needle biopsy specimens whichrequire less invasive sample gathering procedures compared toconventional biopsy specimens. In addition, other immunological methodshave been developed and are now well known in the art which allow fordetection and measurement of cancer markers in non-cellular samples suchas serum and other biological fluids from patients. The use of thesealternative sample sources substantially reduces the morbidity and costsof assays compared to procedures employing conventional biopsy samples,which allows for application of cancer marker assays in early screeningand low risk monitoring programs where invasive biopsy procedures arenot indicated.

For the purpose of breast cancer evaluation, the use of conventional orneedle biopsy samples for cancer marker assays is often undesirable,because a primary goal of such assays is to detect the cancer before itprogresses to a palpable or mammographically detectable tumor stage.Prior to this stage, biopsies are generally contraindicated, makingearly screening and low risk monitoring procedures employing suchsamples untenable. Therefore, there is general need in the art to obtainsamples for breast cancer marker assays by less invasive means thanbiopsy, for example by serum withdrawal.

Efforts to utilize serum samples for breast cancer marker assays havemet with limited success, largely because the targeted markers areeither not detectable in serum, or because telltale changes in thelevels or activity of the markers cannot be monitored in serum. Inaddition, the presence of breast cancer markers in serum probably occursat the time of micro-metastasis, making serum assays less useful fordetecting pre-metastatic disease. In contrast, fluid within the mammaryglands themselves is expected to contain much higher and morebiologically relevant levels of breast cancer markers than serum,particularly in view of the fact that 80%-90% of all breast cancersoccur within the intraductal epithelium of these glands. Fluid withinthe breast ducts is expected to contain an assemblage and concentrationof hormones, growth factors and other potential markers comparable tothose secreted by, or acting upon, the surrounding cells of thealveolar-ductal system. Likewise, mammary fluid is expected to containcells and solid cellular debris or products that can be used incytological or immunological assays to evaluate intracellular or cellsurface markers that may not be detectable in the liquid fraction ofmammary fluid.

Previous attempts to develop non-invasive breast cancer marker assaysutilizing mammary fluid samples have included studies of mammary fluidobtained from patients presenting with spontaneous nipple discharge. Inone of these studies, conducted by Inaji et al., Cancer 60: 3008-3013,1987, levels of the breast cancer marker carcinoembryonic antigen (CEA)were measured using conventional, enzyme linked immunoassay (ELISA) andsandwich-type, monoclonal immunoassay methods.

These methods successfully and reproducibly demonstrated that CEA levelsin spontaneously discharged mammary fluid provide a sensitive indicatorof nonpalpable breast cancer. In a subsequent study, also by Inaji etal., Jpn. J. Clin. Oncol. 19: 373-379, 1989, these results were expandedusing a more sensitive, dry chemistry, dot-immunobinding assay for CEAdetermination. This latter study reported that elevated CEA levelsoccurred in 430 of patients tested with palpable breast tumors, and in73% of patients tested with nonpalpable breast tumors. CEA levels in thedischarged mammary fluid were highly correlated with intratumoral CEAlevels, indicating that the level of CEA expression by breast cancercells is closely reflected in the mammary fluid CEA content. Based onthese results, the authors concluded that immunoassays for CEA inspontaneously discharged mammary fluid are useful for screeningnonpalpable breast cancer.

Although the evaluation of mammary fluid has been shown to be a usefulmethod for screening nonpalpable breast cancer in women who experiencespontaneous nipple discharge, the rarity of this condition renders themethods of Inaji et al, inapplicable to the majority of women who arecandidates for early breast cancer screening. In addition, the firstInaji report cited above determined that certain patients sufferingspontaneous nipple discharge secrete less than 10 μl of mammary fluid,which is a critically low level for the ELISA and sandwich immunoassaysemployed in that study. It is likely that other antibodies used to assayother cancer markers may exhibit even lower sensitivity than theanti-CEA antibodies used by Inaji and coworkers, and may therefore notbe adaptable or sensitive enough to be employed even in dry chemicalimmunoassays of small samples of spontaneously discharged mammary fluid.

In view of the above, an important need exists in the art for morewidely applicable, non-invasive methods and materials to obtainbiological samples for use in evaluating, diagnosing and managing breastdisease including cancer, particularly for screening early stage,nonpalpable breast tumors. A related need exists for methods andmaterials that utilize such readily obtained biological samples toevaluate, diagnose and manage breast disease, particularly by detectingor measuring selected breast cancer markers, or panels of breast cancermarkers, to provide highly specific, cancer prognostic and/ortreatment-related information, and to diagnose and manage pre-cancerousconditions, cancer susceptibility, breast infections and other breastdiseases.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to providenon-invasive methods and kits for obtaining biological samples that canbe employed in assays for evaluating, diagnosing and managing breastdisease, particularly cancer.

It is a further object of the invention to achieve the above object inassay methods and kits that are widely applicable to a broad range ofpatients, and that include useful assays and kits for screening earlystage, nonpalpable mammary tumors.

It is yet another object of the invention to provide methods and kitsthat utilize the aforementioned biological samples to evaluate, diagnoseand manage breast disease, preferably breast cancer, by detecting and/ormeasuring selected breast disease markers such as breast cancer markers,or panels of breast cancer markers, to provide highly specificprognostic and/or treatment-related information to the clinician.

The invention achieves these objects and other objects and advantagesthat will become apparent from the description which follows byproviding non-invasive methods for obtaining biological samples from amammary organ of a mammalian patient. Specifically, the methods of theinvention involve administering oxytocin or an oxytocin analog to amammalian patient in an amount that is effective to stimulate expressionof mammary fluid from a nipple of the patient. The oxytocin ispreferably administered intranasally and is allowed to reach a targetalveolar-ductal tissue of the breast where the oxytocin stimulatesmyoepithelial contraction of the alveolar-ductal tissue. Alternatively,an intramuscular or intravascular injection of oxytocin can effect thesame myoepithelial contraction response as the intranasal administrationroute.

The amount, timing and/or mode of oxytocin administration may beadjusted on an individual basis depending on such factors as menstrualcycle stage, use of birth control or hormone replacement therapy,pregnancy history, age of onset of menarch, enthnicity and other factorsknown to affect an individual's propensity for breast fluid expression.

A mammary fluid collector, preferably a breast pump, is then optionallyapplied to the nipple and is used to receive the expressed breast fluid.In preferred methods involving use of a breast pump, negative pressureis generated on the breast to facilitate the oxytocin stimulatedexpression of mammary fluid. Alternatively, the mammary fluid can beexpressed and collected without the aid of a breast pump, which mayrequire an increase of oxytocin dosage or lengthening of the postadministration time period before breast fluid is fully expressed fromthe nipple.

During or after the mammary fluid expression step, a biological sampleis collected from the expressed mammary fluid, which sample may consistof whole mammary fluid, whole cells, cell fragments, cell membranes,selected liquid, cellular or other solid fractions of the mammary fluid,as well as proteins, glycoproteins, peptides, nucleotides (including DNAand RNA polynucleotides) and other like biochemical and molecularconstituents of the mammary fluid.

Sample collection can be achieved simply by receiving the expressedmammary fluid within any suitable reservoir, such as an ordinary samplestorage container or assay vessel. In preferred embodiments of theinvention, the expressed mammary fluid is exposed to a solid phasesample collection medium, simultaneous with or subsequent to the time ofbreast fluid expression. Suitable solid phase media in this contextinclude microscopic glass slides, capillary tubes, coated tubes,microtiter wells or plates, membranes, filters, affinity columns, dotblot matrices, beads, microspheres, resins, and other like media thatwill selectively adsorb, bind, filter, partition or otherwise processdesired components of the mammary fluid for convenient incorporationinto a desired assay. Often it will be desirable to combine a pluralityof solid phase media for sample collection, eg., a filter and membrane,a membrane and a particulate medium, etc., for example to differentiallypartition and adsorb selected components of the breast fluid. Inconjunction with sample collection, the sample may be exposed to otheragents such as buffers, diluents, extraction or chromatographic media,cross-linking agents, denaturing agents, etc., to stabilize or otherwiseprepare the sample for processing within a desired assay.

Also provided within the invention are methods and devices for obtaininga biological sample from a patient and/or determining the amount of abreast disease marker in a biological sample from breast fluid whichemploy a novel breast pump or breast pump adapter. The breast pumpfunctions in a similar fashion as a conventional breast pump but alsoprovides a solid phase sample collection medium in fluid connection withthe pump. The solid phase sample collection medium may be integratedwithin the breast pump or otherwise fluidly connected therewith, so thata sample of expressed breast fluid contacts the collection medium whilethe pump remains applied to the breast.

In related aspects of the invention, methods are provided fordetermining the presence or amount of a breast disease marker,preferably a breast cancer marker, in biological samples obtained from amammary organ of a mammalian patient. These methods involve intranasal,intramuscular or intravascular administration of oxytocin or an oxytocinanalog to mammalian patients in amounts effective to stimulate mammaryfluid expression in the patient. Once a sufficient post-administrationtime period has elapsed to allow the oxytocin to reach and stimulatetarget alveolar-ductal tissues, mammary fluid is collected directly fromthe nipple or, alternatively, the breast is pumped, and a biologicalsample from expressed mammary fluid is collected, as above. After thesample is collected a bioassay is conducted on the sample to determinethe presence and/or amount of the breast disease marker in the sample.Suitable bioassays in this regard include assays to detect known markersof breast disease, such as assays employing immunological or othersuitable probes to detect specific antigens and other markers expressedby selected pathogens, including bacterial and viral pathogens. Morepreferred bioassays will detect individual markers or panels of markersof benign breast tumors, pre-cancerous breast disease, and/or breastcancer, such as assays employing immunological or other suitable probesto detect specific antigens and other markers expressed by benign,pre-cancerous and/or cancerous alveolar-ductal cells of the breast.Preferably, the assay will detect the presence or amount of multiplebreast disease markers in the biological sample, for example byincluding a panel of immunological or molecular probe(s) that bind orreact with multiple breast cancer markers.

In yet additional aspects of the invention, clinically useful kits areprovided for determining the presence and/or amount of a breast diseasemarker, preferably a breast cancer marker, in biological samplesobtained from a mammary organ of a mammalian patient. The kits include apharmaceutical preparation of oxytocin in a biologically suitablecarrier. Preferably, the oxytocin preparation is a solution of oxytocinprovided in an intranasal spray applicator. The kits also preferablyinclude a collecting device for collecting a biological sample from theexpressed mammary fluid, which collecting device may range from a simplefluid reservoir to solid phase media that can be directly incorporatedinto solid phase bioassays. In this context, an optional breast pump orbreast pump adapter may also be provided serving a dual purpose ofapplying negative pressure to the breast to facilitate mammary fluidexpression from the nipple following oxytocin stimulation, and toprovide a solid phase sample collection medium in fluid connection withthe breast pump for biological sample collection.

In particularly preferred embodiments of the invention, kits includecompositions and/or devices for detecting the presence or amount of oneor more breast disease marker(s) in the biological sample, for exampleone or more immunological or molecular probe(s) that binds or reactswith one or more breast cancer marker(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partial sectional view of a breast pump employing theconcepts of the invention.

FIG. 2 is a sectional view of a portion of the breast pump as indicatedin FIG. 1.

FIG. 3 is a perspective view of a support member for supporting a solidphase sample collection medium in fluid connection with a breast pump.

FIG. 4 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

FIG. 5 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

FIG. 6 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

FIG. 7 is a perspective view of an alternative support member forsupporting a solid phase sample collection medium in fluid connectionwith a breast pump.

FIG. 8 is a sectional view of a breast pump device employing theconcepts of the invention.

FIG. 9 is a sectional view of a portion of a breast pump illustrating asupport member and cartridge for containing a particulate solid phasesample collection medium.

FIG. 10 is a sectional view of a portion of a breast pump illustrating asupport member and an exemplary solid phase sample collection template(coated tube).

FIG. 11 is a partial sectional view of a breast pump employing areciprocating mechanism to adjust positioning of a solid phase samplecollection medium within the pump.

FIG. 12 depicts a breast pump adapter employing the concepts of theinvention.

FIG. 13 depicts a breast pump adapter employing a reciprocatingmechanism to adjust positioning of a solid phase sample collectionmedium within the adapter.

FIGS. 14 and 15 provide partial sectional views of a breast pumpemploying a sliding reciprocating mechanism to adjust positioning of asolid phase sample collection medium within the pump.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the invention provides methods for obtaining biologicalsamples from mammary fluid. Preferably, these methods are non-invasive,meaning they are non-surgical and do not involve penetration of thebreast by needles or other intrusive devices. To achieve a non-invasivesample collecting method, the invention relies specifically onadministering the peptide hormone oxytocin to a mammalian patient, in anamount that is effective to stimulate expression of mammary fluid from anipple of the patient when a breast pump is applied to the nipple toassist the mammary fluid expression. Preferably the oxytocin preparationis administered intranasally and is administered in an amount that isintranasally effective to stimulate expression of mammary fluid from thenipple.

Oxytocin is a peptide hormone of pituitary origin which is naturallyreleased into the bloodstream of lactating women in response tosuckling, and stimulates contraction of myoepithelial cells in themammary alveoli and ducts to cause milk ejection. Cobo. J. Perinat. Med.21: 77-85, 1993. The drug has also been widely used for stimulatinglabor in pregnant women, due to its activity of stimulating uterinecontractions. Satin et al., Am. J. Obstet. Gynecol. 166: 1260-1261,1992. For these reasons, the pharmacology of oxytocin has beenthoroughly investigated, including detailed studies of effectivedosages, half-life and potential side effects.

For use in the present invention, an oxytocin preparation is providedfor intranasal, intramuscular, or intravenous administration thatcontains oxytocin in a biologically suitable, liquid carrier. As usedherein, “oxytocin” refers to natural or synthetic oxytocin andbiologically active derivatives and analogs thereof. Naturally occuringoxytocin from mammalian sources is of course suitable, as are otherknown, naturally occuring oxytocin-like peptide analogues and theirsynthetic counterparts having similar activities for stimulatingalveolar-ductal myoepithelial contraction.

Preferably, the oxytocin used within the invention is a simple peptidehormone comprising a cyclic peptide, the peptide having a well definedring portion (Cys-Tyr-Ile-Gln-Asn-Cys) and tail portion (Pro-Leu-Gly).However, numerous derivatives and analogues are known, or readilyobtainable, in the art, eg., derivatives or analogues having amino acidtruncations, deletions or substitutions at one or more residues of thepeptide and which exhibit substantially the same activity as naturallyoccurring oxytocin (i.e., having at least 75%, and preferably 85%-95% ormore, activity compared to that of native oxytocin for stimulatingalveolar-ductal myoepithelial contraction). The most economic oxytocinpreparations for use within the invention contain a synthetic oxytocin(eg. PITOCIN® or SYNTOCINON® available from various providers, forexample Sandoz (Basel, Switzerland) and United States Pharmacopeia. Foruse in the methods and kits of the invention, a preferred oxytocinpreparation contains approximately 40 USP units of oxytocin per ml ofliquid carrier. Preferred liquid carriers are biologically compatiblesolutions, such as a lactated Ringer's solution or other physiologicallybalanced, sterile, non-toxic and non-irritative solution. To administerthe oxytocin intranasally, a standard nasal squeeze bottle is used,which delivers approximately 0.5 ml of the oxytocin preparation into thepatient's nostril when squeezed. The oxytocin is absorbed by the nasalmucosa into the systemic circulation where it reaches and actsspecifically on the myoepithelial cells surrounding the alveoli of thebreast and making up the walls of the lactiferous ducts, causing theirsmooth muscle fibers to contract and force any fluids present into thelarge ducts or sinuses where it can be expressed from the nipplespontaneously onto a sample collector or by the further action of abreast pump. Intranasal application of the spray preparation istherefore a practical and effective method of administration. Thehalf-life of oxytocin in the human bloodstream is extremely short,estimated to be about 10-15 minutes or less, due to its rapid removalfrom plasma by the kidney, liver, and mammary gland, and the time topharmacokinetic and clinical steady state is readily determineddepending on the mode of administration (eg. bolus dosage, repeatadministration, or steady infusion). (See for example, Gonser, Arch.Gvnecol. Obstet. 256: 63-66, 1995; and Orhue, Obstet. Gynecol. 83:229-233, 1994, each incorporated herein by reference in its entirety).It is therefore a routine matter to determine an appropriateconcentration and dose of the oxytocin preparation to administer aneffective amount (either intranasally effective, intravenouslyeffective, or intramuscularly effective) of the oxytocin to causeexpression of mammary fluid with or without the assistance of a breastpump. (See for example, Newton, Ann. N.Y. Acad. Sci. 652: 481-483; Mena,Neuroendocrinoloqy 61: 722-730, 1995; Gonser, Arch. Gynecol. Obstet.256: 63-66, 1995; Orhue, Obstet. Gynecol. 83: 229-233, 1994; Satin etal., Am. J. Obstet. Gynecol., 166: 1260-1261, 1992; and Satin et al.,Obstet. Gynecol. 83: 234-238, 1994, each incorporated herein byreference in its entirety).

Although not all female patients are expected to be responsive tointranasal oxytocin stimulation, an intranasally effective amount ofoxytocin for the purposes of the invention can be readily determined. Asused herein, an intranasally effective amount of oxytocin is an amountof oxytocin sufficient to intranasally stimulate the expression of atleast 3 μl of mammary fluid in at least 500 of non-lactating femalepatients with the aid of negative pressure to the nipple of between50-200 mm Hg applied by a breast pump up to 45 min after a firstadministration of the oxytocin spray. It may be necessary, and indeedpreferred, to administer a low, preliminary dose of oxytocin to thepatient, for example a single spray of a 40 Unit/ml oxytocin solution ineach nostril, or multiple sprays of a lower concentration oxytocinpreparation, and thereafter wait to determine a particular patient'ssensitivity. If there is no reaction with an initial application of thebreast pump after a short post-administration period of 2-15 minutes,and preferably 2-5 minutes, a booster dose of the oxytocin spray may beadministered and the pump reapplied. In this way, the clinician canmodulate the dosage to each patient's varying sensitivity, and therebyminimize potential adverse side effects. Alternatively, an effectivedose of intramuscular or intravenous oxytocin can be used according tothe same dosage determination and administration principles in patientswhere intranasal administration fails or is otherwise contra-indicatedas a preferred mode of administration.

As noted above, the amount, timing and/or mode of oxytocinadministration may be adjusted based on specific factors known to renderindividuals more or less sensitive to induction of breast fluidexpression. These factors are generally well known in the art, andinclude, for example, menstrual cycle stage, use of birth control orhormone replacement therapy, pregnancy history, age of onset of menarch,and enthnicity, among other factors.

Thus, in one aspect of the invention, methods for obtaining a biologicalsample from a patient and/or determining the amount of a breast diseasemarker in a biological sample from breast fluid are provided whichinclude a step of determining a menstrual stage of the patient. Based onthe determined menstrual stage, a drug administration protocol isselected having a predetermined oxytocin dosage, timing and/or frequencyof oxytocin delivery, and/or mode of oxytocin administration.

According to these methods, one or more variables of oxytocin dosage,timing and/or frequency of oxytocin delivery, and/or mode of oxytocinadministration are selected depending on whether the patient is stagedwithin one of five approximate menstrual phases. These phases include 1)a proliferative phase (characterized by a tight configuration of thealveolar lumena); 2) a follicular phase (characterized by a definedconfiguration of the alveolar lumena); 3) a luteal phase (characterizedby an open configuration of the alveolar lumena, with some secretion bythe alveolar cells); 4) a secretory phase (characterized by an openconfiguration of the alveolar lumena, with secretion by the alveolarcells); and 5) a menstrual phase (characterized by a distendedconfiguration of the alveolar lumena, with secretion by the alveolarcells).

It is generally not desired to conduct the methods of the invention forpatients staged in the proliferative or follicular stage of theirmenstrual cycle (approximately 3-7 days and 8-14 days, respectively).However, in some circumstances sample collection can be performed forthese individuals using high and/or repetitive doses of oxytocin andotherwise optimizing the breast fluid expression response by selecting aparticular mode of oxytocin administration, or combination thereof (eg.,intravenous administration followed by intranasal administration). Forpatients staged in the luteal or secretory stage of their menstrualcycle (approximately 15-20 days and 21-27 days, respectively),intermediate dosages of oxytocin are selected and repetitiveadministrations are reduced or eliminated. For patients staged in themenstrual phase, dosages of oxytocin and repetitive administrations arereduced even further while still providing an effective administrationprotocol to yield sufficient breast fluid expression.

Determination of effective administration protocols for patients ofdifferent menstrual stages can also be readily achieved within theinvention. As used herein, an effective administration protocol yieldsat least 3 μl of expressed mammary fluid in at least 50% ofnon-lactating female patients at an equivalent menstrual stage with theaid of negative pressure to the nipple of between 50-200 mm Hg appliedby a breast pump up to 45 min after a first administration of theoxytocin spray. Various combinations of oxytocin dosage, timing and/orfrequency of oxytocin delivery, and/or mode of oxytocin administrationare contemplated, as can be readily determined by the skilled artisan inaccordance with the teachings herein. Likewise, it will often bepreferred to administer a low, preliminary dose of oxytocin to thepatient and thereafter wait to determine a particular patient'ssensitivity, even when an individual's menstrual stage has beendetermined and a particular administration protocol selected. Thus, ifthere is no reaction with an initial application of the breast pumpafter a short post-administration period, a booster dose of the oxytocinmay be administered and the pump reapplied. In this way also, theclinician can apply a first, stage specific dose of oxytocin andthereafter modulate the dosage, period of time between boosteradministrations, and/or mode of administration, to each patient'svarying sensitivity.

In other, related aspects of the invention, methods for obtaining abiological sample from a patient and/or determining the amount of abreast disease marker in a biological sample from breast fluid areprovided which include a step of determining a non-menstrual stagepatient sensitivity index. Examples of such indices include 1) patientuse of hormone based birth control; 2) patient use of hormonereplacement therapy; 3) patient pregnancy history; 4) patient age ofonset of menarch; and 5) patient enthnicity. Other indices associatedwith sensitivity to induction of breast fluid expression are alsocontemplated. These factors can be determined by such routine steps aspatient consultation, evaluation of patient records, and clinical orlaboratory-based analysis (eg., physical screening, measurement ofsex-steroid hormone levels, etc.) Based on a determined non-menstrualstage sensitivity index, an effective drug administration protocol isselected having a predetermined oxytocin dosage, timing and/or frequencyof oxytocin delivery, and/or mode of oxytocin administration, inaccordance with the methods described above. In yet additional methodsan effective drug administration protocol is selected by firstdetermining both a patient's menstrual stage and at least onenon-menstrual stage sensitivity index specific to the patient, andthereafter selecting an effective oxytocin administration protocol basedon these combined indices.

In yet additional methods within the invention, it may be preferred toconduct the foregoing sample collection methods in conjunction with aconventional mammographic procedure. In this manner, costs, time andpatient discomfort can be minimized. Further, by conducting the samplecollection immediately following a mammogram it is expected that breastfluid expression may be facilitated by breast manipulation during theinitial procedure. Additional steps to facilitate breast fluidexpression include manual breast massage and application of heat packsto the breast.

Once an effective dose(s) of oxytocin is administered and the clinicianhas allowed a suitable post-administration period to elapse for theoxytocin to reach and stimulate the target alveolar-ductal tissue, thebreast pump is applied according to well known procedures and negativepressure is generated on the breast to facilitate the expression ofmammary fluid. Within the methods of the invention, negative pressuresof 50-200 mm Hg are preferred, and these pressures are maintained,preferably intermittently, for approximately 1-15 minutes, depending onthe sensitivity of individual patients, oxytocin dosage and otherfactors. Alternatively, mammary fluid expression can be achieved withoutthe aid of the breast pump using a separate sample collector to receivethe expressed breast fluid, as described herein.

The volume of expressed mammary fluid will vary depending on a varietyof factors, including patient sensitivity to oxytocin, dosage ofoxytocin delivered, time and pressure of breast pump administration, andother factors. For the least sensitive breast marker assays of theinvention, a volume of expressed mammary fluid of 300-500 μl ispreferred to provide ample material for conducting the assay, and thesevolumes will be obtainable from a substantial proportion of womentreated according to the above methods. To express 300-500 μl of mammaryfluid, some women will require repeated stimulation treatments, perhapsrequiring pooling of mammary fluid samples obtained during multiplepatient visits. However, for more sensitive assays of the invention, eg.solid phase immunoassays, much smaller samples of 3 μl or less will besuitable to carry out the assays, particularly in the case of breastcancer markers that are naturally secreted into the mammary fluid andare therefore expected to be present in very high concentrationscompared to, for example, breast epithelial cell surface antigens orintracellular antigens that are not secreted.

During or after the mammary fluid expression step, a biological sampleis collected from the expressed mammary fluid. A range of suitablebiological samples are contemplated and will be useful within themethods of the invention, including whole mammary fluid, selected liquidor solid fractions of the mammary fluid, whole cells or cellularconstituents, proteins, glycoproteins, peptides, nucleotides (includingDNA and RNA polynucleotides) and other like biochemical and molecularconstituents of the mammary fluid. Sample collection can be achievedsimply by receiving the expressed mammary fluid within a suitablereservoir, such as an ordinary sample storage container or assay vessel.

In preferred embodiments of the invention, the expressed mammary fluidis contacted with a solid phase sample collection medium, simultaneouswith or subsequent to the time of breast fluid expression. Suitablesolid phase media in this context include microscopic glass slides,capillary tubes, coated tubes, microtiter wells or plates, membranes,filters, affinity columns, dot blot matrices, beads, resins, and otherlike media that will selectively adsorb, bind, filter, partition orotherwise process desired components of the mammary fluid for convenientincorporation into a desired assay.

A wide range of sample collection procedures and materials known in theart are useful within the invention.

Selected methods and materials will vary among different assays, as willbe understood and readily practiced by those skilled in the art. Forexample, if the breast disease marker sought in a particular assay is asoluble protein, it will often be desired to immobilize the protein on asolid phase matrix or template by contacting the target protein with areagent having high specificity for the protein, preferably a polyclonalor monoclonal antibody. The yields a complex, eg., a ligand-proteincomplex, an antibody-antigen complex, or other complex in which thetarget protein is bound to a specific binding partner (i.e., wherein thecomplex is not dissociated upon addition of a non-specific bindingpartner conventionally used as a control to determine specific binding;and preferably wherein the binding partner binds with an affinity of kD10⁻⁹ or greater). The binding partner which binds to the target proteinis in turn immobilized to the solid phase medium, before or aftercomplex formation with the target protein. Immobilization of the bindingpartner, eg., by covalent binding to a solid phase template or matrix,can be achieved by a variety of conventional methods known in the art.

In this manner, the target protein/binding partner complex is adsorbedor otherwise bound directly to an insoluble matrix. Alternatively, avariety of secondary binding partners, eg., anti-isotype antibodies, maybe added to bind the complex to the insoluble matrix. The latter stepdepends on the nature of the first binding partner (i.e., the bindingagent that specifically binds the target protein), for example whetherthe first binding partner is a primary antibody, ligand, etc.

Particularly useful within the invention are immunoassay which formatsemploy a combination of solid phase or immobilized reagents and labeledreagents whereby the association of the label with the solid phase is afunction of the presence or absence of reactivity with the targetedantigen. In general, such a solid phase reagent comprises a bindingsubstance such as an anti-antibody (eg., anti-IgG), or otherimmunobinder or other binding agent according to the assay protocolinvolved, bound or attached, covalently or noncovalently, to the solidphase matrix or in an otherwise immobilized form.

Useful labeled reagents in solid phase immunoassays include a bindingsubstance such as an anti-antibody (eg., anti-IgG), or otherimmunobinder or other binding agent according to the assay protocolinvolved, which is chemically coupled with a detectable chemical moiety.Useful labels are conventional in the art and include fluorescers,chemiluminescers, radioisotopes, and enzymes. Enzyme labels areparticularly useful and are generally selected from alkalinephosphatase, peroxidase, and ©β-galactosidase.

Enzyme labels are readily detectable by addition of a correspondingchromogenic substrate and detecting the resulting color or fluorescentresponse.

A variation of this protocol uses a ligand-modified form of the targetedantigen(s) with immobilization to the solid phase being accomplished byusing a solid phase bearing an immobilized (eg., bound or adsorbed)binding partner to the ligand. For example, biotin or a hapten (eg.,fluorescein) can be used as the ligand and can be immobilized by contactwith a solid phase form of avidin or anti-hapten antibody, respectively.The addition of the solid phase binding partner can occur at anyconvenient time in the assay, such as prior to contact of sample withthe ligand-antigens(s) or thereafter.

Preferred solid phase matrices for use within the foregoing methodsinclude Staphylococcus aureus or Protein A or G Agarose [eg. Sepharose®(Pharmacia Biotech AB, Uppsala, Sweden)] beads. Protein A and protein Gare cell wall proteins isolated from specific bacterial strains, andhave specific binding sites for certain classes of immunoglobulins.Protein A binds (to varying degrees) most subclasses of IgG, plus IgM,IgA, and IgD. Protein G binds nearly all subclasses of IgG, but notother classes of immunoglobulins.

An alternative solid phase sample collection and/or assay methodutilizes a specific anti-marker primary antibody which is covalentlyattached to the solid phase matrix, eg., by covalent linking theantibody through its free amino groups to cyanogen-bromide-activatedSepharose particles. Insolubilized antibody can be used to pull thecorresponding marker antigen out of solution by adsorption to itssurface. In yet another alternative format, the marker protein can betreated with a cross-linking reagent (eg. biotin or digoxigenin) whichmay be subsequently detected by a second binding partner. In the case ofbiotin, the second binding partner is avidin or streptavidin; fordigoxigenin, the second reagent is an anti-digoxigenin antibody. Avidinand streptavidin may be coupled directly to the solid phase medium, eg.,to agarose beads. Because the initial biotinylation is not specific forthe marker, samples are frequently electrophoresed on, eg., SDS PAGE,transferred to nitrocellulose etc., and Western blotted with antibodiesspecific for the protein factor.

A preferred assay method for detecting protein markers is the wellknown, Enzyme Linked Immunosorbant Assay (ELISA) assay. According tothis method, a variety of coating reagents can be adsorbed or otherwisebound directly onto a surface of a desired solid phase sample collectionmedium, eg., a microtiter plate, well, tube, bead, test strip, plasticmicroparticle, latex particle, etc., to form a coated template ormatrix. These coating reagents are typically a species specificanti-isotype antibody (eg., anti-mouse-IgG) but can also include ananti-marker primary antibody or an affinity reagent such as avidin orstreptavidin. The target protein (eg., a soluble protein marker) iscontacted with a specific primary antibody or, alternatively, iscrosslinked (eg., to biotin) or otherwise modified to form a complex,and the resulting complex is adsorbed to the coated template or matrixand processed according to conventional assay methods.

Latex or particle agglutination methods are also to be mentioned.Particles are coated or covalently coupled with a target antigen,ligand, antibody or other binding partner. The particles are thenincubated with a test sample and resulting agglutination of theparticles, eg., due to formation of ICA antibody linkages betweenparticles, is detected. Detection can be accomplished by visualobservation (eg., a slide agglutination format) or quantified bymeasuring turbidity changes with a spectrophotometer or nephelometer. Awell known variation of this general method based on inhibition ofparticle agglutination can also be employed. In addition, anagglutinator reagent can be prepared comprising multiple antigens, eg.,a water soluble polymer backbone to which are attached multiples of oneor more antigens within a panel.

Alternative methods for collecting and analyzing samples within theinvention include Western immunoblot and dot-blot methods. Forapplication of these methods, the solid phase sample collection mediumis preferably a membrane or filter, eg., a nitrocellulose,polyvinylidene difluoride (PVDF), or nylon membrane. Proteins within thebreast fluid sample may be processed (eg., separated on SDS PAGE) ordirectly transferred to the membrane, and non-specific interactions maybe blocked by incubating the membrane with, eg., bovine serumalbumin/ovalbumin or non-fat dry milk. A primary antibody withspecificity for the protein marker is contacted with the membrane, andexcess antibody is washed, eg., with buffered detergent. A labelledisotype specific antibody is next contacted with the membrane, andtarget protein-primary antibody-secondary antibody ternary complexes aredetected, eg., calorimetrically.

Where the targeted protein factor includes a carbohydrate moiety, thefactor can also be adsorbed to a solid phase template or matrix, eg., aresin, by way of lectin-carbohydrate interactions. Various lectins areavailable for this purpose which differ in their carbohydrate bindingspecificity. For example, Lectin Con A binds to mannose-containingcarbohydrate structures and with low affinity to α-glucose andα-N-acetylglucosamine. Lectin GNA binds to terminal mannose residues.Lectin MAA binds to α(2-3) Linked sialic acids. A variety of otherlectins collectively providing a wide range of specificities are knownin the art.

A particularly preferred solid phase sample collection medium for usewithin the invention is a filter, pad or membrane that can be directlycontacted to a sample of expressed breast fluid to adsorb, absorb, bind,partition or otherwise facilitate sample processing or handling within aselected assay. For this purpose, several types of transfer membranesare known, including nitrocellulose which is the most commonly usedtransfer membrane. Several commercial sources now offer nitrocelluloseimpregnated with a synthetic support which improves its durability andflexibility without altering its performance. One preferred transfermembrane, polyvinylidene difluoride (PVDF), marketed by Millipore(Bedford, Mass.) under the trade name IMMOBILON®, has slightly lowerprotein-binding capacity than nitrocellulose but is mechanicallystronger and compatible with many organic solvents. This allows directprotein staining with Coomassie Blue, and direct amino acid compositionand sequence analysis of transferred proteins, without interfering withits subsequent use for antibody probing.

Membranes are not only useful within the invention for protein blotting,but also for immobilization of nucleic acids. Thus, nitrocellulose,reinforced nitrocellulose, diazotized membranes (paper or nylon), nylon,charged nylon, or PVDF, and DEAE-anion exchange membranes are useful forimmobilizing DNA and RNA from expressed breast fluid. In this context,the most commonly used membranes are reinforced nitrocellulose andnylon. Nitrocellulose has a lower background but also a lower bindingcapacity than nylon and is chosen primarily when background, but notdetectability, is the main concern. Nylon, in contrast, is ideal forlower copy number sequences, short target sequences (down to oligomers)or for reprobing. Membranes are also available with different poresizes. For DNA blots, membranes with a pore size of 0.45 μm are usuallychosen for large fragments, but 0.22 μm for fragments of <500 bases. ForRNA blots, membranes with a pore size of 0.1 or 0.22 μm are mostefficient. Membranes are available in different size specifications,including sheets, rolls, pre-cut circles, etc.

Methods for detecting DNA on nylon without DNA purification andprocessing of the samples, eg., for detecting DNA from fluids or wholecells, have recently been developed (Reed and Matthaei, Nucleic AcidsRes. 18: 3093 (1990; Hammermueller et al., J. Virol. Methods 31: 47(1991), each incorporated herein by reference). These procedures avoidenzymatic dispersion of cells, RNase and pronase treatments to hydrolyzecellular macro-molecules, etc., and are typically based on the capacityof alkali and other reagents to disperse and solubilize cells andhydrolyze macro-molecules including RNA and protein, but not DNA.Positively charged modified nylon membranes then irreversibly bindnucleic acid while remaining suitable for hybridization.

Nucleic acid extraction and processing steps may also be minimized bywell known fast blot methods. In particular, fast blot methods which usenylon as a solid phase take advantage of the ability of NaOH todissociate cells, denature DNA and immobilize DNA. Nitrocellulosemembranes have a lower binding capacity and co-immobilization of nucleicacid and protein from neutral solutions can be a problem. ConcentratedNaI can be used to inhibit protein immobilization, to denature DNA andto irreversibly bind the nucleic acid to nitrocellulose without arequirement for baking. This method can also be used for RNA.

Although it is possible to directly transfer proteins, nucleic acids andother markers to a solid phase matrix which is in turn directlyincorporated in an assay, it may be desirable to concentrate the targetmarker, eg., by chromatography, extraction, specific or nonspecificadsorption, etc., particularly when sensitivity is a problem. Thus,samples can be collected and initially processed by contacting breastfluid with a solid phase chromatographic medium, eg., within a cartridgecomprising a micro-column of Sepharose-coupled antibody. Up to 500-foldincreases in immunoassay sensitivity with apparent recoveries of 85 to95% can be achieved using this approach. This and other well knownchromatographic procedures provide a powerful approach to thequantitation of substances too dilute to be measured by routine methods.

For sample collection and processing using chromatographic and relatedmethods, a particulate solid phase sample collection medium ispreferred. Various particulate media are known which selectively adsorb,absorb, bind, or partition components of biological samples, which mediaare readily adapted for collection and processing of breast fluidsamples. These particulate can be coupled with various coating reagentsknown in the art, eg., affinity reagents, to provided a coated medium,or may be used in an unmodified form.

Exemplary particulate sample collection media for use within theinvention include beads, plastic microparticles, latex microspheres,glass materials such as controlled porous glass, granular agarose basedmaterials, cross-linked dextran polymers, inorganic or organic ionexchanger materials, kieselsur and other silicate materials. Suitablematerials additionally include cellulosic materials, eg.,diethylaminoethyl (DEAE) cellulose or diethylamino (DEA) cellulose. Alsouseful are natural polymeric carbohydrates and their syntheticallymodified, cross-linked or substituted derivatives, such as agar, agaroseand cross-linked dextran polymers.

Synthetic polymers which can be prepared with suitably porousstructures, such as vinyl polymers (eg., polyethylene, polypropylene,polystyrene, polyvinylchloride, polyvinylacetate and its partiallyhydrolysed derivatives, polyacrylates, polyacrylamides,polymethacrylates), copolymers and terpolymers of the above vinylmonomers among themselves and with other monomers, polycondensates (eg.,polyesters and polyamides), and addition polymers, such as polyurethanesor polyepoxides are also useful.

Yet additional particulate media are prepared from inorganic materialshaving a suitably porous form, such as sulfates or carbonates ofalkaline earth metals and magnesium. Examples include barium sulfate,calcium sulfate, calcium carbonate, magnesium carbonate, silicates ofalkali and alkaline earth metals and/or aluminum and/or magnesium, andaluminum or silicon oxides or hydrates, such as clays, alumina, talc,kaolin, zeolite, among others.

Also included among useful solid phase sample collection media porousbarrier materials suitable for use with breast pump and breast pumpadapter devices of the invention, for example to enclose particulatesolid phase media within a cartridge adapted for coupling in fluidconnection with a breast pump or breast pump adapter. Such porousbarrier materials are inert to and nonreactive with markers and otheranalytes and reagents used in assaying for breast disease markers, andare porous with respect to the passage of liquids and/or particulates ofa pre-selected size. Suitable materials include various porous materialssuch as nylon fabric, polyethylene and other plastic films, membranes,filters, glass wool, sponge, styrofoam, ceramic and other porousmaterials.

In conjunction with sample collection, samples of expressed breast fluidmay be exposed to other agents such as buffers, diluents, extraction orchromatographic media, cross-linking agents, blocking agents, denaturingagents, etc., to stabilize or otherwise prepare the sample forprocessing within a desired assay. For example, the sample may bediluted (eg., by collecting the sample in a well or recess containingthe solid phase medium wetted or suspended in a diluent) to minimizenonspecific binding effects, eg., affecting a subsequent immunoassay. Inthe exemplary context of sample collection for immunoassays, the avidityof the antibody for the marker antigen is an important consideration,whereby providing more or less diluent during sample collection andincubation may optimize a particular antigen-antibody system beingstudied.

Commonly used buffers for dilution include phosphate, borate, orTris-buffered saline. Usually, the choice of the buffer is notimportant. Nonetheless, a careful examination of the effect of buffer,pH, ionic strength, and divalent cations will facilitate use of a newsample collection/assay system in order to maximize sensitivity andresolve possible sources of interference within the assay. Althoughimmunoassays are usually carried out at neutrality, doing so is notalways optimal.

Nonspecific binding or adsorption, eg., of antigens and haptens(especially hydrophobic haptens) to glass and plastic tubes or pipetsmay markedly influence measured activity in a particular immunoassay.With some proteins and polypeptides, nonspecific binding in immunoassaysis reduced if plastic tubes are used. The addition of protein to themedium may also minimize nonspecific adsorption and help avoiddenaturation of highly diluted antigens and antibodies. Therefore,assays involving iodinated antigens are generally carried out inprotein-containing buffers. Bovine serum albumin, gelatin, lysozyme, andovalbumin are commonly used, usually at final concentrations of 1 to 5mg/ml. In some systems diluted whole serum or proteins present in thesample itself are just as satisfactory. However, even though addedproteins are often beneficial, they should not be used indiscriminatelywithout making an evaluation for possible adverse effects, for examplecontaminating enzymes that may degrade the marker protein.

Other possible additives for improved sample collection and assaymethods, apart from buffer and protein, include enzyme inhibitors andchelating agents. In assays lasting longer than 3 days, a bacteriostaticagent, such as sodium azide, 0.1 to 0.2%, may also be incorporated intothe sample collection and/or assay medium to help avoid microbialgrowth.

Although a fundamental utility of the present invention lies in thenovel, non-invasive methods for obtaining biological samples frommammary fluid, additional methods are disclosed herein that provideuseful assays for detecting and/or measuring important breast diseasemarkers in these samples. In this context, the invention provides abroad range of assay methods incorporating known procedures and reagentsfor determining the presence and/or expression levels of breast diseasemarkers, particularly breast cancer markers, in biological samples. Asincorporated within the invention, these methods involve administrationof oxytocin to mammalian patients, preferably via intranasaladministration, in amounts effective to stimulate mammary fluidexpression in the patient, as described above. Once a sufficientpost-administration time period has elapsed to allow the oxytocin toreach and stimulate target alveolar-ductal tissues, the breast is pumpedand a biological sample is collected, as described above. After thesample is collected, a bioassay is conducted on the sample to determinethe presence and/or amount of a selected breast disease marker,preferably a breast cancer marker or panel of breast cancer markers, inthe sample.

As used herein, the term breast disease marker refers to any cell, cellfragment, protein, peptide, glycoprotein, lipid, glycolipid,proteolipid, or other molecular or biological material that is uniquelyexpressed (eg. as a cell surface or secreted protein) by diseased breastcells, or is expressed at a statistically significant, measurablyincreased or decreased level by diseased breast cells, or in associationwith breast disease (eg. a protein expressed by an infectious agentassociated with breast disease), or is expressed at a statisticallysignificant, measurably increased or decreased level by diseased breastcells compared to normal breast cells, or which is expressed bynon-diseased breast cells in association with breast disease (eg. inresponse to the presence of diseased breast cells or substances producedtherefrom). Breast disease markers can also include specific DNA or RNAsequences marking a deleterious genetic change, or an alteration inpatterns or levels of gene expression significantly associated withbreast disease. Preferred breast disease markers include markers ofbreast infections, benign neoplasia, malignant neoplasia, pre-cancerousconditions, and conditions associated with an increased risk of cancer.

As used herein, the term breast cancer marker refers to a subset ofbreast disease markers, namely any protein, peptide, glycoprotein,lipid, glycolipid, proteolipid, or other molecular or biologicalmaterial that is uniquely expressed (eg. as a cell surface or secretedprotein) by cancerous cells, or is expressed at a statisticallysignificant, measurably increased or decreased level by cancerous cellscompared to normal cells, or which is expressed by non-cancerous cellsin association with cancer (eg. in response to the presence of cancerouscells or substances produced therefrom). Breast cancer markers can alsoinclude specific DNA or RNA sequences marking a deleterious geneticchange, or an alteration in patterns or levels of gene expressionsignificantly associated with cancer. In addition, breast cancer markerscan include cytological features of whole cells present in mammaryfluid, such as nuclear inclusions or cytoplasmic structures or stainingattributes uniquely expressed by, or associated with, cancerous cells.

Among the breast cancer markers that are useful within the methods ofthe invention, a subset are described in representative review articlesby Porter-Jordan et al., Hematol. Oncol. Clin. North Amer. 8: 73-100,1994; and Greiner, Pharmaceutical Tech, May, 1993, pp. 28-44, eachincorporated herein by reference in its entirety. Other suitable markersare also widely known and can be readily incorporated into the methodsof the invention using information and methods generally known oravailable in the literature. Preferred breast cancer markers for usewithin the invention include well characterized markers that have beenshown to have important value for determining prognostic and/ortreatment-related variables in human female patients. As notedpreviously, prognostic variables are those variables that serve topredict outcome of disease, such as the likelihood or timing of relapseor survival. Treatment-related variables predict the likelihood ofsuccess or failure of a given therapeutic program. Determining thepresence or level of expression or activity of one or more of thesemarkers can aid in the differential diagnosis of patients with malignantand benign abnormalities, and can be useful for predicting the risk offuture relapse or the likelihood of response to a selected therapeuticoption.

It is important to note, however, that the invention does not relysolely on breast disease markers that meet the stringent requirements ofsensitivity and specificity that would render the marker immediatelyacceptable for clinical application to human patients. On the contrary,a number of breast disease markers contemplated within the inventionfall short of these stringent criteria, and nonetheless provide usefulinformation that can be of substantial benefit in detecting,differentially diagnosing or managing breast cancer. Such non-clinicallyaccepted markers are useful for immediate application within the methodsof the invention as basic research tools, and as adjunctive tools inclinical applications. Beyond these immediate applications, many suchmarkers are expected to be further developed and refined according tothe methods of the invention to the point of direct clinicalapplicability, particularly in assay methods that analyze combinationsof markers to generate complementary data of greater predictive valuethan data yielded by individual markers alone.

The preferred assay methods of the invention particularly focus onbreast cancer markers associated with tumorigenesis, tumor growth,neovascularization and cancer invasion, and which by virtue of thisassociation provide important information concerning the risk, presence,status or future behavior of cancer in a patient. As noted previously,tumorigenesis and tumor growth can be assessed using a variety of cellproliferation markers (for example Ki67, cyclin D1 and PCNA). Tumorgrowth can also be evaluated using a variety of growth factor andhormone markers (for example estrogen, EGF, erbB-2, and TGF-U, receptorsof autocrine or exocrine growth factors and hormones (for example IGFand EGF receptors), or angiogenic factors. In addition to tumorigenic,proliferation and growth markers, a number of markers provideinformation concerning cancer invasion or metastatic potential in cancercells, for example by indicating changes in the expression or activityof cell adhesion or motility factors. Exemplary markers in this contextinclude Cathepsin D, plasminogen activators and collagenases. Inaddition, expression levels of several putative tumor “suppressor”genes, including nm23, p53 and rb, provide important data concerningmetastatic potential, or growth regulation of cancer cells. A largenumber and variety of suitable breast cancer markers in each of theseclasses have been identified, and many of these have been shown to haveimportant value for determining prognostic and/or treatment-relatedvariables relating to breast cancer.

Prior to or concurrent with each assay run of the invention, it may bepreferable to perform a preliminary evaluation to verify sample originand/or quality. The focus of such preliminary evaluations is to verifythat the sample collected from expressed mammary fluid is indeed ofmammary origin, and is not contaminated with other potentialcontaminants, such as sweat from skin surrounding the nipple. For thesesample verification purposes, a variety of assays are available whichidentify mammary fluid markers known to be present in mammalian mammaryfluid, and which are preferably highly specific markers for mammaryfluid (i.e. markers which are typically always present in mammary fluidand which are absent from all, or most of, other potentiallycontaminating bodily fluids and tissues). However, an acceptable levelof specificity for mammary fluid markers within the methods of theinvention is provided by markers that are simply known to be present inmammary fluid, even though they may be present in other bodily fluids.One such marker is the enzyme lysozyme, which is a normal component ofhuman serum, urine, saliva, tears, nasal secretions, vaginal secretions,seminal fluid, and mammary fluid. Lysozyme (muramidase) is an enzymewhich hydrolyzes beta 1,4-glycosidic linkages in the mucopolysaccharidecell wall of a variety of microorganisms resulting in cell lysis.Quantitative measurement of lysozyme is readily accomplished by a wellknown agar plate diffusion method, described in detail in theinstructions provided with the QUANTIPLATE® lysozyme test kit, availablefrom Kallestad, Sanofi Diagnostics (Chasta, Minn.), incorporated hereinby reference in its entirety.

Other mammary fluid markers for sample verification that are morespecific than lysozyme are preferred within the methods of theinvention, and can be readily incorporated within the invention based onpublished and generally known information. The most preferred amongthese markers are proteins and other biological substances that arespecifically expressed or enriched in mammary fluid. A diverse array ofsuitable markers in this context have been characterized and havealready been used to develop specific antibodies, including affinitypurified and monoclonal antibodies. These antibodies can in turn beemployed as immunological probes to determine the presence or absence,and/or to quantify, selected mammary fluid markers to verify mammaryfluid sample origin and quality. Mammary fluid markers of particularinterest for use within the invention include specific cytokeratins thatare characteristically expressed by normal and cancerous mammaryepithelial cells, against which specific panels of antibody probes havealready been developed. (See for example, Nagle, J. Histochem. Cytochem.34: 869-881, 1986, incorporated herein by reference in its entirety).Also useful as mammary fluid markers are the human mammary epithelialantigens (HME-Ags) corresponding to glycoprotein components of the humanmilk fat globulin (HMFG) protein, against which specific antibodies(eg., anti HMFG1, Unipath, U.K.) are also available. (See Rosner et al.,Cancer Invest. 13: 573-582, 1995; Ceriani et al. Proc. Natl. Acad. Sci.USA 74: 582-586, 1982; Ceriani et al., Breast Cancer Res. Treat. 15;161-174, 1990, each incorporated herein by reference in D its entirety).

To conduct the breast disease marker assays provided within theinvention, a collected biological sample from mammary fluid is generallyexposed to a probe that specifically binds to a selected breast diseaseor breast 3 cancer marker, or otherwise interacts with the marker in adetectable manner to indicate the presence or absence, or amount, of thebreast disease or breast cancer marker in the sample. Selected probesfor this purpose will generally depend on the characteristics of thebreast disease marker, i.e. on whether the marker is a proteinpolynucleotide or other substance. In preferred embodiments of theinvention, the breast disease marker is a protein, peptide orglycoprotein, all of which are effectively targeted in breast diseasemarker assays using specific immunological probes. These immunologicalprobes can be labeled with a covalently bound label to provide a signalfor detecting the probe, or can be indirectly labeled, for example by alabeled secondary antibody that binds the immunological probe to providea detectable signal.

General methods for the production of non-human antisera or monoclonalantibodies (eg., murine, lagormorpha, porcine, equine) are well knownand may be accomplished by, for example, immunizing an animal with aselected breast disease marker protein, peptides synthesized to includepart of the marker protein sequence, degradation products including partof the marker protein sequence, or fusion proteins including all or partof the marker protein linked to a heterologous protein or peptide.Within various embodiments, monoclonal antibody producing cells areobtained from immunized animals, immortalized and screened, or screenedfirst for the production of an antibody that binds to the selectedbreast cancer marker protein or peptide, and then immortalized. It maybe desirable to transfer the antigen binding regions (i.e., F(ab′)2 orhypervariable regions) of non-human antibodies into the framework of ahuman antibody by recombinant DNA techniques to produce a substantiallyhuman molecule. Methods for producing such “humanized” molecules aregenerally well known and described in, for example, U.S. Pat. No.4,816,397 (incorporated herein by reference in its entirety).Alternatively, a human monoclonal antibody or portions thereof may beidentified by first screening a human B-cell cDNA library for DNAmolecules that encode antibodies that specifically bind to the selectedbreast disease marker according to the method generally set forth byHuse et al. (Science 246: 1275-1281, 1989 (incorporated herein byreference in its entirety). The DNA molecule may then be cloned andamplified to obtain sequences that encode the antibody (or bindingdomain) of the desired specificity.

Also contemplated within the invention are bifunctional antibodieshaving independent antigen binding sites on each immunoglobulin molecule(as disclosed for example in Thromb. Res. Suppl. X: 83, 1990, and in TheSecond Annual IBC International Conference on Antibody Engineering, A.George ed., Dec. 16-18, 1991; each incorporated herein by reference inits entirety), as well as panels of individual antibodies havingdiffering specificities. Bifunctional antibodies and antibody panels ofparticular use within the invention include antibodies and panels ofantibodies that bind to two or more selected breast disease markers togenerate complementary data of greater predictive value than datayielded by individual markers alone.

Monoclonal antibodies are particularly useful within the invention aslabeled probes to detect, image and/or quantify the presence or activityof selected breast disease markers. In this context, monoclonalantibodies that specifically bind to selected breast disease markers areprovided which incorporate one or more well known labels, such as a dye,fluorescent tag or radiolabel. By incorporating such a label, theantibodies can be employed in routine assays to determine expression,localization and/or activity of one or more selected breast diseasemarkers in a biological sample including, or derived from, mammaryfluid. Results of these assays to determine expression, localizationand/or activity of a selected breast disease marker in a test sampletaken from a patient at risk for breast disease, or known to have breastdisease, can be compared to results from control studies detectingand/or quantifying the same marker in biological samples obtained fromnormal patients negative for breast disease. In this manner, baselinedata and cutoff values can be determined according to routine methods torefine the assays of the invention and adapt them for direct clinicalapplication.

Detection and/or quantification of breast disease markers in thebiological samples of the invention can be accomplished using a varietyof methods. Preferred methods in this regard include well known ELISAimmunoassays, immunoprecipitation assays, and various solid phaseimmunoassays including Western blotting, dot blotting and affinitypurification immunoassays, among other methods. Comparable methods aredisclosed herein, or are elsewhere disclosed and known in the art, forusing non-antibody probes to detect and/or quantify the expressionand/or activity of breast disease markers. Suitable non-antibody probesfor use within the invention include, for example, labeled nucleotideprobes that hybridize at moderate or high stringency to DNA transcriptsof oncogenes and other DNA sequences associated with elevated breastdisease risk, or with mRNA transcripts encoding breast disease markerproteins. Preferably, the nucleotide probes hybridize with a targetsequence under high stringency conditions. As used herein, “moderatestringency” and high stringency” refers to finite ranges ofhybridization conditions that are well established in the literature.(See, for example: Sambrook et al., (1989) Molecular Cloning ALaboratory Manual (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press);Hames and Higgins, eds., Nucleic Acid Hybridization A PracticalApproach, IRL Press, Washington D.C., 1985; Berger and Kimmel, eds,Methods in Enzymology, Vol. 52, Guide to Molecular Cloning Techniques,Academic Press Inc., New York, NY, 1987; and Bothwell, Yancopoulos andAlt, eds, Methods for Cloning and Analysis of Eukaryotic Genes, Jonesand Bartlett Publishers, Boston, MA 1990; each of which is incorporatedherein by reference in its entirety. Moderate or high stringencyhybridization conditions are achieved, eg., by adjusting the temperatureof hybridization, adjusting the percentage of helix-destabilizing agentssuch as formamide in the hybridization mix, and adjusting thetemperature and salt concentration of the wash solutions. Alternatively,stringency can be adjusted during post-hybridization washes by varyingthe salt concentration and/or the temperature. Stringency ofhybridization may be reduced by reducing the percentage of formamide inthe hybridization solution or by decreasing the temperature of the washsolution. Typical high stringency conditions require, for example, hightemperature hybridization (eg., 65-68° C. in aqueous solution containing4-6×SSC, or 42° C. in 50% formamide) combined with a high temperature(eg., 5-25° C. below the To) wash and a low salt concentration (eg.,0.1×SSC). In contrast, moderate stringency conditions involve, forexample, hybridization at a temperature between 50° C. and 55° C. andwashes in 0.1×SSC, 0.1% SDS at between 50° C. and 55° C., which shouldbe sufficient to identify polynucleotide molecules encoding I-mf fromother species or to isolate isoforms of I-mf. In further contrast, lowstringency conditions involve, for example, low hybridizationtemperatures (eg., 35-42° C. in 20-50% formamide) and intermediatetemperature (eg., 40-60° C.) washes in a higher salt concentration (eg.,2-6×SSC).

In certain preferred embodiments of the invention, cDNA andoligonucleotide probes are employed in well known Northern, Southern anddot-blot assays for identifying and quantifying the level of expressionof a selected breast disease marker in cell samples collected fromexpressed mammary fluid.

Other suitable probes for use within the invention include labeledligands, binding partners and co-factors of breast disease markers (eg.growth factor receptor ligands, or substrates of breast cancerassociated proteases such as cathepsin D).

Measuring the level of expression of breast disease markers according tothe foregoing methods will provide important prognostic andtreatment-related information for assessing a broad range of breastdisease, including the genesis, growth and invasiveness of cancer, inmammals, particularly humans. For example, assays utilizingoligonucleotide probes will assist early screening to evaluate heritablegenetic lesions associated with breast cancer, and to distinguishbetween pre-cancerous, early cancerous and likely metastatic lesions inpatients.

In addition to the above mentioned sample collection and assay methods,the invention also provides kits and multicontainer units comprisingreagents and components for practicing the sample collection and assaymethods of the invention. Briefly, these kits include basic componentsfor obtaining a biological sample from mammary fluid, including apharmaceutical preparation of oxytocin in a biologically suitablecarrier. Preferably, the oxytocin preparation is provided in anintranasal spray applicator and contains approximately 40 USP units ofoxytocin per ml of liquid carrier, which carrier is a simple,inexpensive buffered saline solution. Preferred applicators can be inany of a variety of pressurized aerosol or hand-pump reservoir forms,with a nozzle for directing a liquid spray of the oxytocin into apatient's nostril. The kits also preferably include a collecting devicefor collecting a biological sample from the expressed mammary fluid,which collecting device may range from a simple fluid reservoir to solidphase media that can be directly incorporated into solid phasebioassays. In this context, an optional breast pump may also be providedthat is applicable to a human breast and designed to generateintermittent or sustained negative pressures in an area surrounding thenipple of between about 50-200 mm Hg. More preferably, the breast pumpserves a dual purpose of applying negative pressure to the breast tofacilitate mammary fluid expression from the nipple following oxytocinstimulation, and to provide a reservoir or solid phase collecting deviceincorporated within the breast pump for biological sample collection.

Kits for practicing the assay methods of the invention include asuitable container or other device for collecting a biological samplefrom expressed mammary fluid. A range of suitable collection devices arecontemplated corresponding to a wide range of suitable biologicalsamples that may be collected from the expressed mammary fluid. Forexample, simple sterile containers or reservoirs are provided to collectwhole mammary fluid. Alternatively, a variety of solid phase devices,including microscopic glass slides, membranes, filters, beads and likemedia, are provided to receive or partition selected liquid or solidfractions of the mammary fluid, to receive or partition cells orcellular constituents from the mammary fluid, or to receive or partitionpurified or bulk proteins, glycoproteins, peptides, nucleotides(including DNA and RNA polynucleotides) or other like biochemical andmolecular constituents from the mammary fluid. A wide variety of suchsample collection devices are disclosed herein, or are otherwise widelyknown or described in the literature, which can be readily adapted foruse within specific embodiments of the invention. These collectiondevices may be provided as a component of the breast pump (such as aremovable nitrocellulose filter placed within the pump to directlyreceive or contact the expressed mammary fluid as it is pumped), or maybe provided separately (for example as a non-integral membrane, filter,affinity column or blotting material to which mammary fluid or mammaryfluid components are exposed to collect a biological sample for assaypurposes).

In more detailed embodiments of the invention, kits include reagentsand/or devices for detecting the presence and/or amount of a breastdisease marker in the biological sample, for example an immunological ormolecular probe that binds or reacts with a breast cancer marker. Amongthese possible reagents or devices are immunological andnon-immunological probes for detecting the presence or amount of abreast cancer marker in the biological sample. The kits may also containsuitable buffers, preservatives such as protease inhibitors, direct orsandwich-type labels for labeling the probes, and/or developing reagentsfor detecting a signal from the label. In one aspect, kits of thepresent invention contain monoclonal antibodies useful for detectingand/or measuring a breast cancer marker in a sample. Such antibodies maybe pre-labeled, or may be detected by binding to a secondary antibodyoptionally included in the kit. The antibody reagents may be provided ina separate container, or may be provided in combination in a series ofcontainers. Within yet another aspect of the invention, kits containsequence-specific oligonucleotide primers for detecting polynucleotidemolecules encoding breast cancer marker proteins. Such primers may beprovided in separate containers, or may be provided in combinations ofone or more primer pairs in a series of containers. A broad selection ofother kits are provided within the invention based on general knowledgein the art and on the description herein, including kits that containspecific instructions for carrying out the assays of the invention.

Also provided within the invention are methods for obtaining abiological sample from a patient and/or determining the amount of abreast disease marker in a biological sample from breast fluid, whichmethods employ a novel breast pump 10 or breast pump adapter 12, asdescribed hereinbelow. These methods include a step of applying thebreast pump to assist breast fluid expression, wherein a solid phasesample collection medium is fluidly connected with the breast pump. Thesolid phase sample collection medium may be integrated within the breastpump or otherwise fluidly connected with the pump, so that an expressedbreast fluid sample contacts the collection medium while the pumpremains applied to the breast.

To practice these aspects of the invention, the breast pump 10 (FIG. 1)and breast pump adapter 12 (FIG. 11) each have fluidly connectedtherewith a solid phase sample collection medium selected from any ofthe solid phase media described hereinabove. The breast pump may begenerally constructed according to various conventional breast pumpdesigns, for example according to the general design described in U.S.Pat. No. 4,929,229 and U.S. Pat. No. 5,007,899 to Larsson; U.S. Pat. No.5,601,531 to Silver; U.S. Pat. No. 3,786,801 to Sartorius; or U.S. Pat.No. 5,295,957 to Aida et al.

As with other conventional breast pumps, the breast pump of theinvention includes a breast engaging portion 14 constructed of anon-porous material. The engaging portion is sized and dimensioned toreceive at least a nipple 16 portion of a breast 17 and form a suctionseal therewith. Preferably, the breast engaging portion is sized anddimensioned to receive at least an areolar portion of the breast, andmore preferably a distal quarter to one-half or larger portion of thebreast (eg., as shown in FIG. 1), and form a suction seal therewith.Different sizes and dimension of the breast engaging member may beselected, eg., to receive human breasts of differing sizes.Alternatively, devices for veterinary use are provided wherein thebreast engaging member is sized and dimensioned to receive a breast of anon-human mammal.

To form a suction seal with the breast 17 as described above, the breastengaging portion 14 of the pump 10 may be constructed in a variety ofshapes and dimensions. In one embodiment the engaging portion is formedas a simple cylinder, tube or funnel shaped and dimensioned to engagethe nipple 16 or areolar portion of the breast in a suction seal.Preferably, a terminal edge 18 of the engaging portion is rounded orflared so that the edge does not impinge uncomfortably against the skinof breast 17 when negative pressure is applied to the breast to form thesuction seal. In preferred embodiments the engaging portion is roughlyfunnel shaped to comfortably engage a distal quarter to one half orlarger portion of the breast, as shown in FIG. 1 and form a suction sealtherewith.

The breast engaging portion 14 of the breast pump 10 can be constructedof any suitable non-porous material which is inert to body fluids andwhich has sufficient rigidity to prevent collapse of the engagingportion when negative pressure is applied against its inner walls 20.Preferably, the engaging portion and other parts of the breast pump areautoclavable for sterilization purposes. Thus, the engaging portion maybe constructed of a rigid material such as a polypropylene,polyurethane, polyvinyl plastic, polymethyl methacrylate, and the like.Alternatively, the engaging portion may be constructed of a semi-rigidmaterial which prevents collapse but allows for manual compression of atleast a base 22 of the engaging portion to massage the nipple 16 and/orareolar region of the breast 17 to facilitate breast fluid expression.Suitable materials in this context include rubber or syntheticelastomers, eg., silicon plastic (silastic) and like materials.Preferably, the material which forms the engaging portion is transparentto allow a physician or technician using the breast pump to visualizethe breast 17 to determine its positioning and condition duringapplication of the pump and to observe fluid expression from the nipple.

The breast engaging portion 14 of the breast pump 10 is fluidlyconnected to a sample collection housing 30 made of a rigid material(preferably transparent plastic) which supports a solid phase samplecollection medium in fluid connection with the engaging portion. In oneaspect of the invention the housing supports a pad, or sheet, 38 ofabsorbent or adsorbent material, for example a membrane 39 or filter 40pad or sheet (FIGS. 2-5). Multiple pads or sheets (of the same ordifferent material) may be used in combination. For example, a membrane39 (eg., nitrocellulose) may be supported on a filter 40 (eg., a paperfilter) as shown in FIG. 5. In this manner, a first sheet may serve as asupport member, a wetting member, a wicking member, or a partitioningmember for a second sheet, or may introduce or remove a chemicalreagent, probe, blocking agent, buffering agent, denaturing agent, etc.therefrom. In one aspect, the multiple sheet materials partitioncomponents of the breast fluid (eg., by using different materials toretain different components of the breast fluid), thereby allowing forcollection of different samples simultaneously.

In another aspect of the invention the housing supports a particulatesolid phase sample collection medium 41, for example beads, resins,microspheres, particulate chromatographic media (eg., agarose orsilicate media), and the like (see, eg., FIG. 9). In yet another aspectof the invention, the housing supports a non-particulate solid templatefor sample collection, for example one or more capillary tubes 42 (FIG.6), coated tubes 43 (FIG. 10), plates, wells, slides and the like formedof glass, plastic or other suitable materials.

As shown in FIGS. 1 and 2, a preferred design of the breast pump 10includes a removable coupling mechanism between the engaging portion 14and the sample collection housing 30. A preferred coupling mechanismincludes complementary threads 44, 46, disposed at mated connecting ends48, 50 of the engaging portion, and housing, respectively.Alternatively, a simple pressure fit coupling may be provided toremovably couple mated connecting ends 48, 50 of the engaging portionand housing, as shown in FIG. 9. In yet another alternative embodiment,the connecting ends 48, 50 are removably coupled by a hinge 52 and latch54 that pivotally connects the two connecting ends (FIG. 10).

The sample collection housing 30 can support the solid phase samplecollection medium in several ways, as exemplified in the drawings andalso using a variety of equivalent designs that will be apparent to theartisan. In preferred embodiments of the invention, the solid phasemedium is held on or within a support member 56 adapted to be fixedlyinterposed between the engaging portion 14 of the breast pump 10 and thehousing.

Thus, in one exemplary design shown in FIGS. 1 and 2, the support member56 is a removable disc spanning a lumen 58 of the housing and interposedbetween connecting ends 48, 50 of the engaging portion and housing. Foruse in conjunction with a variety of breast pump designs, a diameter 59(FIG. 3) of the support member is between about ¼-3.0 inches, preferablyabout ½-2.0 inches, and more preferably about ¾-1 inches. In preferredaspects, the disc-shaped support member seats within a circumferentialgroove 60 in the connecting end 48 of the housing. A complementarycircumferential groove 62 in the connecting end 46 of the engagingportion opposes the circumferential groove in the connecting end of thehousing to sandwich the disc-shaped support member therebetween.

Prior to connecting the engaging portion 14 of the breast pump 30 withthe housing 30, the support member is seated therebetween (eg. byfitting the support member within the opposing circumferential grooves60, 62 of the housing and engaging portion). The force of connection(i.e. threading, pivoting or pushing the engaging portion and housingrelative to one another) firmly sandwiches the support member inposition between the engaging portion and housing.

To facilitate this purpose, the thickness (i.e., sectional height) 63 ofthe support member 56 is equal to or slightly greater than the height ofa sidewall 64 of the circumferential groove 60 of the housing 30,whereby the support member is held in a friction fit and may bepartially compressed when the engaging portion and housing areconnected. Thus, the thickness of the support member is between about 2mm to 5 cm, preferably about 3 mm to 2 cm, and more preferably about 4mm to 1 cm. Consistent with this design, the support member can be madeof a hard plastic material (eg., a hard polyvinyl or polyurethane), butis preferably made of a resilient, moderately compressible material,eg., soft plastic, rubber, or a waterproof fiber or composite materialas used in conventional plumbing and automotive gaskets.

A disc-shaped support member 56 is well suited to support a sheet 38 ofabsorbent or adsorbent material, such as a membrane or filter. As shownin FIGS. 2 and 7, the sheet is preferably sandwiched between an upperretainer ring 66 and a lower retainer ring 68 of the support member tohold the sheet in place against negative pressure that may pass throughthe filter when a vacuum is applied through the engaging portion 14 andhousing 30 (see below), as well as when the nipple 16 impinges againstthe sheet. The upper and lower retainer rings may be integrally joinedin a disposable refill as shown in FIG. 2, or the two retainer rings maybe separable to provide a reusable cassette for removing and insertingreplacement sheets. An example of the latter design is depicted in FIG.7, where the upper and lower retainer rings are releasablyinterconnected, eg., by a hinge 71 or other connecting means such as aninterlocking threading or detent fit mechanism. In this embodiment theupper and lower rings can be opened or disconnected to allow insertionand removal of the sheet, and juxtaposingly closed, eg., by a snap 72 onone ring adapted to form a detent fit within a receptacle 74 on theopposing ring, thereby holding the sheet in a fixed position between thetwo rings. To facilitate this purpose, opposing faces 75 of the upperand lower rings may have a rugose or otherwise decorated surface tofacilitate retention of the sheet, for example a ridge 76 or ridges toengage the sheet and securely clamp the sheet between the two rings.

In an alternative design depicted in FIGS. 4 and 5, there is no upperretaining ring 72 and the sheet 38 simply rests upon the support member56 or is removably retained against an upper surface 76 of the supportmember by alternative retaining means. For example, the sheet may befitted within a recess 78 surrounding the upper surface of the supportmember that is shaped and dimensioned to receive the sheet. The sheetmay be securely fitted within the recess, eg., by appropriately sizingthe sheet so an edge of the sheet frictionally engages a sidewall 79 ofthe recess. Alternatively, a retaining groove may be provided betweenthe sidewall of the recess and the upper surface of the support memberto receive the edge of the sheet and thereby retain the sheet by adetention fit within the recess during use. In yet another alternativedesign, the sheet simply rests atop the upper surface of the supportmember and is removably secured thereto, eg., by wetting or gluing(preferably with an inert bonding agent) to create a temporary bondbetween the sheet and upper support member surface. In each of theforegoing designs, the sheet can be easily seated within or atop thehousing for sample collection and removed thereafter for processing,eg., by hand or using forceps or other conventional handling tools.

In preferred embodiments of the invention, the support member 56includes a recess 78 which forms a fluid-retaining well, as shown inFIG. 5. The recess can thus be filled with a desired solution, such as abuffer, a solution containing a probe, cross-linking agent, blockingagent, denaturing agent, etc., to facilitate sample collection,handling, and/or processing.

Where the design of the support member 56 is such that it spans thelumen 58 of the sample collection housing 30, or when the support membercontains a recess 78 forming a well, it is generally desirable toprovide air channels 80 in the support member 56 to allow negativevacuum pressure to pass from the housing through the air channels to theengaging portion 14 of the pump during operation, and to allow ventingof the engaging portion and housing to permit disengagement of theengaging portion from the breast 17 after use. Preferably, one or moresuch air channels are located near the periphery of the support member,as shown in FIGS. 2, 3, 5, 6 and 9. Alternatively, one or more airchannels may be centrally located, as shown in FIG. 4. The air channelsmay be positioned so that they do not communicate with the solid phasesample collection medium, as shown in FIGS. 2, 3, 5, 6 and 9, or theymay communicate and form a gaseous connection therewith (provided thatthe solid phase medium is porous and has sufficient strength towithstand vacuum pressures transmitted through the air channel), asshown in FIG. 4.

Alternative designs and configurations of the housing 30 and/or supportmember 56 are also provided which vary with the type of solid phasesample collection medium used. For example, when a particulate solidphase sample collection medium 41 (eg. beads, resins, or microspheres)is used, the medium may be enclosed in a cartridge 82 removably mountedto, or integrated within, the support member or otherwise removablyconnected to the sample collection housing 30. As shown in FIG. 9,preferred embodiments of the invention provide a removable engagementmechanism which allows the cartridge or other receptacle containing thesolid phase medium to be removably engaged relative to the housing, eg.,by engaging the cartridge with a support member so that a first end ofthe cartridge makes a fluid connection with the engaging portion 14 ofthe pump 10. In one embodiment, the first end of the cartridge isremovably inserted through a mounting channel 86 which passes throughthe support member to provide a fluid connection between the engagingportion of the pump and the cartridge first end. Preferably, the channelis dimensioned to receive the first end of the cartridge in a frictionfit (eg., wherein a diameter of the channel is about 0.5 mm to 2 cm,preferably about 1 mm to 1 cm, and more preferably about 3-5 mm),whereby the cartridge can simply be pushed into the channel until thecartridge first end is flush with, or extends slightly above, the uppersurface 76 of the support member and will remain in place during use.For this purpose it is also preferable to form at least the channelportion of the support member from a resilient, moderately compressiblematerial so that the channel yieldingly receives and releases thecartridge in a moderate (i.e., readily hand removed) friction fit.Alternatively, the cartridge can be engaged relative to the housing bycomplementary threading or interlocking detent fitting (eg., aconventional key and groove design) between the cartridge first end andthe support member channel). In yet other alternative designs thecartridge can be permanently engaged with the support member or engageddirectly to the housing.

Design and construction of the cartridge 82 will vary depending on thecharacteristics of the particulate solid phase medium used, includingthe size of the particles, the function of the particles (eg.,chromatography adsorption, affinity binding, etc.), and whether theparticles are used dry or are contained in a solution, among otherfactors. Design and construction of the cartridge will further depend onthe type of breast disease marker(s) which may be sought for detectionin the sample (eg., cells, proteins, lipids or nucleic acids).

In a preferred embodiment shown in FIG. 9, the cartridge is cylindricaland contains beads or microspheres. To enclose the beads or microspheresin the cylinder while maintaining a fluid connection with the engagingportion 14 of the pump 10, the first end 84 of the cylinder is coveredby a semi-permeable cover 90 of a porous barrier material (eg., a filteror membrane) which allows breast fluid (including or excluding selectedcomponents of the fluid, such as cells) to pass through the cover tocontact the beads or microspheres, while preventing escape of the beadsor microspheres from the cartridge. In this manner, the cover canpartition components of the breast fluid into the cartridge, and canalso separately retain different components on the cover, therebyallowing for collection of different samples simultaneously. Thesemi-permeable cover can be affixed to the cartridge by a variety ofmeans, eg., by bonding with a removable or permanent bonding agent, orby providing a removable or integral cover retaining ring 92 to securethe cover to the cartridge first end 84. A second end of the cartridgefeatures a second end cover 96 which may be integral to or removablefrom the cartridge, and which may be impermeable to gas and fluids orsemi-permeable as described above for the first end cover. In anotheraspect of the invention, the housing 30 supports a non-particulate solidtemplate for sample collection. This type of solid phase collectionmedium includes, eg., one or more capillary tubes 42 (FIG. 6), coatedtubes 43 (FIG. 10), plates, wells, slides and the like. These templatesfor receiving, adsorbing or binding a sample of breast fluid (or desiredcomponents thereof) are preferably formed of glass, plastic or likematerials known in the art to be suitable for sample collection (eg.,inert plastics).

To accommodate these various templates, yet additional alternativedesigns and configurations of the housing 30 and/or support member 56are provided. For example, when capillary tubes 42 are used, these maybe mounted to or integrated within the support member, or anchored by avariety of other comparable means with respect to the housing 30. Asshown in FIG. 6, preferred embodiments of the invention utilize asupport member with one or more mounting channels 86 to removablyreceive a first end of one or more capillary tubes 42, so that the endof the tube makes a fluid connection with the engaging portion 14 of thepump 10. Thus, the channels have a preferred diameter equal to orslightly less than a diameter of a standard capillary tube, i.e., about0.5 mm to 3 mm, preferably about 1-2 mm and more preferably about 1.5mm. Construction of the support member and mounting of the tube(s) issimilar to support member construction and mounting of the cartridge asdescribed above. When a single tube is used, it is preferably placedcentrally relative to the housing. When multiple tubes are used they maybe arrayed to collect multiple samples simultaneously, eg., as shown inFIG. 6.

Another alternative solid template for sample collection provided withinthe invention is a coated tube 43 which is preferably mounted relativeto the housing 30 in the same manner as described above for capillarytubes 42 (FIG. 10). The tube may be open at both ends, or may have asemipermeable cover at one or both ends, as well as an impermeablesecond end cover, as described above for the cartridge 82.

The coated tube has a lumenal coating 100 adapted for adsorbing,binding, partitioning or otherwise processing the breast fluid sample.For example, the coating may be an affinity coating having an antibody,ligand, or other binding partner that specifically binds a selectedbreast disease marker, wherein the coating is covalently or otherwisebound to a lumenal wall of the tube. A wide variety of useful coatingsare disclosed herein or are otherwise well known in the art. Thesecoatings may also be used to coat other solid phase media for use withinthe invention, including templates such as wells, plates, slides, etc,including a well formed by a recess 78 in a support member 56.

Because only small droplets of breast fluid will typically be expressedat the surface of the nipple 16, it is generally preferred to directlycontact the expressed fluid on the nipple with the solid phase samplecollection medium. This requires positioning of the sample collectionmedium close to the base 22 of the breast engaging portion 14 of thepump as shown in the figures. Thus, when a support member 56 is providedit is positioned so that its upper surface 76 will directly contact thenipple during application of negative pressure through the engagingportion to the breast. Only approximate positioning is generallyrequired in this regard, because the nipple will tend to be drawn towardthe support member by the vacuum and thereby will abut the upper supportmember surface.

However, because breast pump designs and breast anatomy varysignificantly, it is preferable to adjustably mount the solid phasemedium relative to the housing 30 so that it can be moved closer to, orfarther away from, the base 22 of the engaging portion 14 of the pump10. Thus, in preferred embodiments of the invention a reciprocatingmechanism is provided which adjustably moves the solid phase collectionmedium in closer, or more distant, proximity to the nipple when thebreast pump is engaged therewith. At the beginning of the fluidexpression procedure, the collection medium is retracted away from thenipple while negative pressure is applied to the breast to facilitatefluid expression. Fluid expression is visualized through a transparentengaging portion or housing, and the collection medium is then advancedproximal to the nipple to contact the expressed fluid.

As shown in FIG. 11, a preferred design for the reciprocating mechanismincorporates a support member 56 to support the solid phase collectionmedium, as described above. The support member is reciprocatinglymounted relative to a rotating member 109 of the housing 30, preferablyon a reciprocating carrier 110. The support member may be removablymounted to the carrier, eg., by friction fitting, detention fitting orthreadedly engaging the support member to a first end 112 of thecarrier, as described above for mounting the support member to thehousing and/or engaging portion 14 of the pump 10. For example, thesupport member may be mounted by friction fitting within acircumferential groove 114 at the first end of the carrier. Inconjunction with this design, the carrier is preferably in the form ofan open cylinder so that negative pressure can be effectivelytransmitted through the carrier and support member to the engagingportion.

To reciprocatingly adjust the position of the carrier 110 and/or supportmember 56 relative to the engaging portion 14 of the pump 10, therotating member 109 of the housing 30 is sealably, rotatably, andremovably interconnected to the base 22 of the engaging portion. Thisinterconnection may be accomplished by a variety of designs, one ofwhich is to seat a first O-ring 116 in opposing circumferential grooves118, 120 in the connecting ends 48, 50 of the engaging portion, and therotating member of the housing, respectively. These grooves are sizedand dimensioned to receive the O-ring in an airtight seal when vacuumpressure is applied through the housing and engaging portion of thepump, without substantially compressing the O-ring. The O-ring is alsolubricated, eg., with silicon grease. These features allow free rotationof the rotating member of the housing relative to the engaging portionof the pump, which rotation drives the reciprocating mechanism toadvance the sample collection medium (eg., by advancing the carrierand/or support member) to contact the expressed breast fluid on thenipple 16.

To complete the reciprocating mechanism for the above describedembodiment of the invention, the rotating member 109 of the housing 30is also sealably and rotatably interconnected to a stationary member 124of the housing. This interconnection is preferably achieved by seating asecond O-ring 126 in opposing circumferential grooves 128, 130 in a rearconnecting end 132 of the rotating member of the housing and a frontconnecting end 134 of the stationary member 124 of the housing,respectively. These grooves are also sized and dimensioned to receivethe O-ring in an airtight seal without substantially compressing theO-ring, and the O-ring is lubricated to facilitate free rotation of therotating member relative to the stationary member.

To reciprocate the carrier 110 and/or support member 56 forward andbackward relative to the engaging portion 14, the rotating member 109 ofthe housing 30 is provided with a lumenal, helically oriented groove 140dimensioned to receive a riding peg 142 extending transversely from thecarrier or support member. In addition, the rotating member of thehousing is provided with a longitudinally oriented, lumenal groove 144dimensioned to receive an angularly fixating keel 146 extendingtransversely from the carrier or support member. In accordance with thisdesign, rotation of the rotating member 109 of the housing 30 drivesrotation of the carrier or support member which is angularly fixedrelative to the rotating member by the fixating keel engaged with thelongitudinal groove of the rotating member. As the rotating member ofthe housing and carrier thus rotate (with the position of the engagingportion and stationary member of the housing angularly fixed by frictionor manual or structural resistance), the riding peg rides along thehelical groove, translating the peg in the direction of the groove andthereby causing the support member or carrier to reciprocate forward orbackward relative to the engaging portion.

To insert and remove the solid phase medium and/or support member 56from the rotating member 109 of the housing 30, a removableinterconnection is provided between the rotating member and the base 22of the engaging portion, as described above. To uncouple the rotatingmember and engaging portion, all that is required is that these parts bepulled in opposing directions, whereby the O-ring 116 will unseat fromone of the opposing circumferential grooves 118, 120 in the connectingends 48, 50 of the engaging portion and rotating member, respectively.To recouple the rotating member and engaging portion after loading orretrieval of the sample collection medium and/or support member, theyare simply pushed back together. To facilitate reseating of the O-ring,it may be desired to make one of the opposing circumferential groovesdeeper than the other, so that the deeper groove retains the O-ring whenthe rotating member and engaging portion are separated, and theshallower groove more readily accepts the O-ring when they arere-coupled.

An alternative reciprocating mechanism is provided within the inventionwhich uses a simple slide mechanism to reciprocate the sample collectionmedium relative to the engaging portion 14 of the pump 10, as shown inFIGS. 14 and 15. One embodiment of the slide mechanism features amanifold 150 defining an inner lumen 152 which is not in gaseousconnection with an outer lumen 154 of the housing. This design providesfor a manual slide lever 156 to extend to the outside of the housing sothat a head portion 158 of the lever can be manually engaged by a pumpoperator. The slide lever is in turn connected to the support member 56or carrier 110 which are sized and dimensioned to allow the carrier toreciprocate freely within the inner lumen.

In operation, the slide lever 156 is moved to a rearward position sothat the solid phase sample collection medium (eg., a pad or sheet 38 ofabsorbent material) is out of contact with the nipple 16, as shown inFIG. 14. Negative pressure is applied through the outer lumen 154 to thearea of the breast surrounding the nipple, the tip of which is alignedwith the inner lumen. Breast fluid expression is visualized through thetransparent engaging portion and housing, at which time the lever ismanually engaged by the head portion 158 and moved forward. Movement ofthe lever causes the support member and/or carrier to move forward untilthe sample collection medium contacts the expressed fluid at the tip ofthe nipple. The engaging portion and housing are removably connected,eg., by a hinge 52 and latch 54 or other suitable connection means,thereby allowing for easy insertion and removal of the solid phasemedium and/or support member.

In each of the foregoing breast pump designs, the engaging portion 14 ofthe breast pump 10 is in gaseous connection with a vacuum pump 160capable of generating sustained negative pressure in an area of thebreast 17 surrounding the nipple 16 (see FIG. 1). Any of a large varietyof vacuum pumps, which are well known for use in conjunction with breastpumps, can be used, including manual pumps (FIG. 1), mechanically drivenpumps and electrically driven pumps. When activated, the pump generatesnegative pressures of between about 50-200 mm Hg. Typically the pumpwill be connected via a heavy vacuum hose 162 in connection with theengaging portion. Generally, the hose is connected to the housing 30which will is in gaseous connection with the engaging portion (see, eg.,FIGS. 1, 8 and 11).

Pressure exerted upon the breast 17 by the pump can be varied inaccordance with well known pressure modulating mechanisms (eg., byproviding a diaphragm or other mechanism to modulate a diameter of an inline, pressure modulating valve). In addition, the breast pump 10includes a venting mechanism, eg., a pressure release valve 164, whichthe user can selectively operate to close and vent the system before andafter use, thereby selectively applying and releasing the vacuumpressure acting on the breast. In this regard, the system is generallyvented as soon as sufficient breast fluid expression is observed by theoperator. This also relieves pressure on seals (eg., Q-rings 116, 126),when the reciprocating mechanism relies on a sealable and rotatableconnection between different parts of the pump (as in FIGS. 11 and 13),thereby facilitating respective rotation of the different parts toreciprocate the support member 56 and/or carrier 110.

In yet another aspect of the invention, a breast pump adapter 12 isprovided which couples a solid phase sample collection medium with aconventional breast pump (See FIGS. 12 and 13). As shown in FIG. 12, theadapter features a replacement breast engaging portion 170 sized anddimensioned for removable insertion within a breast engaging portion 14of a conventional breast pump. In preferred embodiments, the replacementbreast engaging portion is funnel shaped and nests within a funnelshaped breast engaging portion of an existing breast pump. When fullynested, a terminal edge 172 of the replacement breast engaging portionextends at least as far as the terminal edge 18 of the breast engagingportion of the existing breast pump.

The replacement breast engaging portion 170 can be removably connectedto the breast engaging portion 14 of the existing breast pump 10 by avariety of means, eg., by friction fitting, detention fitting orthreadedly engaging the replacement engaging portion with the breastengaging portion of the existing pump. Preferably, the adapter 12 has astem portion 174 which extends into a cylindrical, connecting portion176 of the existing pump, and the stem portion cooperates with this partof the existing pump to provide a removable connection mechanism. Thus,in one preferred embodiment the stem portion features a circumferentialgroove 178 dimensioned to receive an O-ring 180, which O-ring impingesagainst an inner wall 182 of the connecting portion to create a frictionfit to interconnect the replacement engaging portion with the breastengaging portion of the existing pump.

The adapter 12 supports a solid phase sample collection medium in fluidconnection with the replacement engaging portion 170. Preferably, thesolid phase medium is connected with the replacement engaging portion bya support member 56, as described above. The support member may beintegrally or removably mounted to the adapter, eg., by frictionfitting, detention fitting or threadedly engaging the support member tothe stem 174 of the replacement engaging portion, in a position thatwill allow contact between the nipple and solid phase medium during orafter breast fluid expression. For example, the support member may bemounted by friction fitting within a circumferential groove 184 at abase of the stem (FIG. 12).

In preferred embodiments of the breast pump adapter 12, a reciprocatingmechanism is provided to move the solid phase sample collection mediumrelative to the replacement engaging portion 172, in accordance with theconcepts described above. As shown in FIG. 13, a preferred design forthe adapter having a reciprocating mechanism features a replacementbreast engaging portion 170 sealably and rotatably nested within arotating dial member 190, which is in turn sized and dimensioned forremovable insertion within a breast engaging portion 14 of an existingbreast pump. The replacement breast engaging portion and rotating dialmember are preferably funnel shaped to collectively nest within a funnelshaped breast engaging portion of a conventional breast pump. When fullynested, a terminal edge 172 of the replacement breast engaging portionand free edge 192 of the rotating dial member extend at least as far asthe terminal edge 18 of the breast engaging portion of the existingbreast pump.

The rotating dial member 172 is connected to a rotating member 109 ofthe housing 30, preferably as a unitary insert, whereby manual rotationof the dial member drives rotation of the rotating member of thehousing. The rotating member of the housing is in turn rotatably coupledwith an anchoring member 194 of the housing which anchors the entirehousing within the existing pump, eg., within a cylindrical, connectingportion 176 of the existing pump. As shown in FIG. 13, the anchoringmember of the housing is preferably in the form of a sleeve whichpartially surrounds the rotating member of the housing and is sealably,rotatably connected therewith. The anchoring member is in turnnon-rotatingly anchored within the cylindrical, connecting portion ofthe existing pump.

In one preferred embodiment the rotating member 109 of the housing issealably, rotatably connected with the anchoring member 194 of thehousing by seating a first Q-ring 196 in opposing circumferentialgrooves 198, 200 at front connecting ends 202, 204 of the rotatingmember and the anchoring member 194 of the housing, respectively. Thesegrooves are sized and dimensioned to receive the Q-ring in an airtightseal between the rotating member and anchoring member, withoutsubstantially compressing the O-ring. The O-ring is also lubricated tofacilitate free rotation of the rotating member relative to theanchoring member. A second, lubricated and non-compressingly seatedO-ring 206 is seated in opposing circumferential grooves 208, 210 atrear connecting ends 212, 214 of the rotating member and anchoringmember of the housing, respectively, to facilitate rotation of therotating member relative to the anchoring member.

To align and facilitate rotation of the rotating member 109 of thehousing, the rotating dial member 190 (which drives the rotating memberof the housing) is sealably, rotatably connected with the replacementengaging portion 170 of the adapter 12. Preferably, the replacementengaging portion has a stem 216 which nests within a stem-shaped base218 of the rotating dial member. Free rotation between these structuresis achieved, eg., by providing a third lubricated and non-compressinglyseated O-ring 220 seated in opposing circumferential grooves 222, 224 inthe stem and base of the replacement engaging portion and rotating dialmember, respectively. This rotation is also facilitated by frictioncontact (by pressure and/or suction) between the replacement engagingportion and the breast 17 of the patient, which angularly secures thereplacement engaging portion and prevents its co-rotation with therotating dial member.

The anchoring member 194 of the housing is in turn anchored within theexisting pump by an anchoring mechanism which angularly secures theanchoring member within the pump, eg., against an inner wall 182 of thecylindrical connecting portion 176. For example, front and rearcompressible anchoring sleeves 230, 232 may be mounted in front and rearcircumferential anchoring sleeve retainer grooves 234, 236 surroundingthe anchoring member. The anchoring sleeves are non-lubricated and aremade of a semi-compressible material such as rubber or soft plastic.This construction creates a friction anchor between the anchoring memberand the inner wall of the connecting portion, so that the anchoringmember does not move angularly during rotation of the rotating member109 of the housing. Both the anchoring sleeves and retainer grooves arepreferably sharply angled at a position corresponding to the bases ofthe retainer grooves (i.e., they have a rectangular or triangularcross-section), to securely retain the anchoring sleeves in the groovesdespite strong friction against the inner wall of the connecting portionwhen the anchoring member of the housing is being inserted into theconnecting portion of the existing breast pump 10 to assemble theadapter 12 with the pump.

Because the replacement engaging portion 170 is anchored by frictionagainst the breast 17, and the anchoring member 194 of the housing 30 isanchored by friction against the inner wall 182 of the connectingportion 176 of the existing pump 10, the rotating member 109 of thehousing rotates freely with respect to both the replacement engagingportion and the anchoring member when an operator manually engages therotating dial member 190 and turns it gently while maintaining pressureagainst the breast.

Relative rotation between the rotating member and anchoring member ofthe housing drives the reciprocating mechanism within the instantembodiment of the invention to advance the sample collection medium(eg., by advancing a carrier 110 and/or support member 56 supporting themedium) toward the replacement engaging portion 170 to contact theexpressed breast fluid on the nipple 16. As with previously describedembodiments, the housing 30 preferably houses a support member 56 tosupport the solid phase collection medium, as described above. Thesupport member is reciprocatingly mounted relative to the anchoringmember 194 of the housing 30, preferably on a reciprocating carrier 110.The support member may be removably mounted to the carrier, eg., byfriction fitting, detention fitting or threadedly engaging the supportmember to a first end 112 of the carrier, as described above. In theembodiment shown in FIG. 13, the support member is removably engagedwith the carrier by cooperative threading 140 between the support memberand carrier. In addition, the support member may be sized anddimensioned for receipt within the stem 216 of the replacement engagingportion, because the replacement engaging portion and an inner (i.e.,lumenal) diameter of the stem thereof are smaller than respectivedimensions of the original engaging portion 14 and its base 22, so thatthe nipple may not fully extend through the stem to contact thecollection medium within the housing. Also in conjunction with thisdesign, the carrier is preferably in the form of an open cylinder andthe rotating member 109 of the housing has a vacuum port 242 so thatnegative pressure can be effectively transmitted through the rotatingmember and carrier (and/or through air channels 80 of the supportmember) to the replacement engaging portion.

To reciprocatingly adjust the position of the carrier 110 and/or supportmember 56 relative to the replacement engaging portion 170 of theadapter 12, the anchoring member 194 of the housing is provided with alumenal, helically oriented groove 140 dimensioned to receive a ridingpeg 142 extending transversely from the carrier or support member. Inaddition, the rotating member of the housing is provided with alongitudinally oriented, lumenal groove 144 dimensioned to receive anangularly fixating keel 146 extending transversely from the carrier orsupport member. Lastly, the rotating member is provided with a second,longitudinally oriented, lumenal groove 244 to allow access of theriding peg through the wall of the rotating member of the housing intothe helically oriented groove and to allow reciprocating passage of thepin along the groove.

In accordance with this design, rotation of the rotating dial member 190drives rotation of both the rotating member 109 of the housing 30 aswell as the carrier 110 (or support member) which is angularly fixedrelative to the rotating member by the fixating keel 146 engaged withthe longitudinal groove 144 of the rotating member. As the rotatingmember and carrier thus rotate (with the position of the replacementengaging portion 170 and anchoring member 194 angularly fixed byfriction or manual or structural resistance), the riding peg rides alongthe helical groove 140, translating the peg in the direction of thegroove and thereby causing the support member or carrier to reciprocateforward or backward relative to the replacement engaging portion.

To insert and remove the solid phase medium and/or support member 56from the adapter 12, removable connections can be uncoupled between theexisting pump 10 and the entire adapter unit, between the rotatingmember 190 and anchoring member 194 of the housing, or between therotating dial member and replacement engaging portion 170, among otheraccess designs which will be readily apparent to those skilled in theart.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE 1

Stimulation of Mammary Fluid Expression by Intranasal Administration ofOxytocin Coupled With Breast Pump Application

Oxytocin nasal solution, acts specifically on the myoepithelial elementssurrounding the alveoli of the breast and making up the walls of thelactiferous ducts, causing their smooth muscle fibers to contract andthus force any fluids present into the large ducts or sinuses where itcan be expressed from the nipple by the further action of a breast pump.The nasal spray is promptly absorbed by the nasal mucosa to enter thesystemic circulation. Intranasal application of the spray preparation isa practical and effective method of administration. Half-life ofoxytocin in the human circulation is extremely short, approximately10-15 minutes, and oxytocin is then rapidly removed from plasma by thekidney, liver, and mammary gland.

Because of the known effects of oxytocin to cause uterine contractions,pregnant women should not be treated by the methods contained hereinunless the benefits of testing outweigh the risk of inducing prematurelabor.

The oxytocin nasal solution contains a concentration of natural orsynthetic oxytocin, or a functional analog thereof, that is intranasallyeffective in a selected volume of administered spray to stimulateexpression of mammary fluid from a nipple of the patient when a breastpump is applied to the nipple to assist mammary fluid expression. In thepresent example, a preferred oxytocin preparation containingapproximately 40 USP units of oxytocin per ml of lactated Ringer'ssolution is administered into the nose with the squeeze bottle held inthe upright position while the patient is in a sitting position. One ortwo sprays are administered into each nostril from a standard nasalsqueeze bottle, which delivers approximately 0.5 ml of the oxytocinsolution per spray in a fine mist when the bottle is squeezed. Thenumber and volume of sprays administered, as well as the concentrationof oxytocin in the solution, can be adjusted according to well knownpharmacokinetic principles (See for example, Newton, Ann. N.Y. Acad.Sci. 652: 481-483; Mena, Neuroendocrinology 61: 722-730, 1995; Gonser,Arch. Gynecol. Obstet. 256: 63-66, 1995; Orhue, Obstet. Gynecol. 83:229-233, 1994; Satin et al., Am. J. Obstet. Gynecol., 166: 1260-1261,1992; and Satin et al., Obstet. Gynecol. 83: 234-238, 1994, eachincorporated herein by reference in its entirety) to ensure that theamount of oxytocin administered to the patient corresponds to anintranasally effective amount to stimulate the expression of at least 3μl of mammary fluid in at least 50% of non-lactating female patientswith the aid of the breast pump. For example, adjustments may be desiredin the is number of sprays delivered to the patient, and/or the timingof spray delivery, so that the clinician can modulate the dosage to eachpatient's varying sensitivity, and thereby minimize potential adverseside effects. In the present example, a preliminary dose of a singlespray of the 40 Unit/ml oxytocin solution is delivered into each nostrilof the patient, and the administering clinician waits for a shortpost-administration period of 2-3 minutes. After this period the breastpump is applied, and the clinician determines whether or not the amountof oxytocin delivered was sufficient to stimulate breast pump assistedexpression of mammary fluid. If no fluid is expressed at this stage abooster dose of 1 or 2 additional sprays of the oxytocin solution isadministered in each nostril, and the pump is reapplied after a 5-10minute post-booster administration period.

After the intranasally effective dose of the oxytocin is administeredand the clinician has allowed a suitable post-adminsitration period toelapse for the oxytocin to reach and stimulate the targetalveolar-ductal tissue, the breast pump is applied according to wellknown procedures. Negative pressures of 50-200 mm Hg are applied in thearea of the nipple and are maintained, intermittently or continuously,for approximately 1-15 minutes, depending on the sensitivity ofindividual patients, oxytocin dosage and other factors. Alternatively,oxytocin can be administered by intramuscular or intravascular routes bywell known means (Oxytocin Injection (synthetic), USP; Wyeth-AyerstLaboratories) to effect the same response as intranasal administration.

Using the above methods, primary samples of mammary fluid containing atleast 3 μl of fluid are expressed by 50% or more of non-lactating femalepatients. During or after the mammary fluid expression step, abiological sample is collected from the expressed mammary fluid. In thepresent example, a nitrocellulose filter is placed within the breastpump in line with a path of the expressed mammary fluid into the pump,so that the expressed fluid contacts the filter. Upon contact of theprimary sample of expressed mammary fluid with the filter, cells,proteins and other desired components of the mammary fluid adhere to thefilter to form a filter-bound biological sample for subsequent analysis.Other suitable biological samples, including whole mammary fluidsamples, cytological samples of whole cells, membranes or other cellularcomponents, and samples containing proteins, glycoproteins, peptides,nucleotides and other constituents of the primary mammary fluid samplecan be collected with appropriate modifications of the above procedures,according to well known principles and methods.

EXAMPLE 2

Verification of Sample Origin and Ouality Using Lysozyme Analysis

To ascertain that the primary sample of mammary fluid, or the collectedsample, obtained by the above methods is of mammary origin and is notcorrupted by likely contaminants, one or more constituents of normalmammary fluid are assayed for. In the present example, an enzyme that isordinarily present in mammary fluid, lysozyme, is assayed in the primarymammary fluid sample to help confirm that the sample is of mammaryorigin. Lysozyme (muramidase) is an enzyme which hydrolyzes beta1,4-glycosidic linkages in the mucopolysaccharide cell wall of a varietyof microorganisms, which activity can be readily detected and quantifiedusing a routine, inexpensive assay. In the present example, Lysozyme ismeasured in the primary mammary fluid sample using the QuantiplateLysozyme Test kit (Kallestad, Chasta, Minn.). The assay employs thereagents and procedures provided by the manufacturer and specified indetail in the manufacturer's instructions, with the exception that amammary fluid sample is substituted in place of serum, urine or tears.Analysis of these results establishes that the sample contains lysozyme,which is a normal component of human serum, urine, saliva, tears, nasalsecretions, vaginal secretions, seminal fluid, and mammary fluid.

More specific assays are used in place of the lysozyme assay, or tosupplement lysozyme assay results, particularly where clinical data forhuman patients are being gathered. Other mammary fluid markers forsample verification that are more specific than lysozyme can be readilyincorporated within the invention, based on published and generallyknown information. In one example, the presence of cathepsin D isassayed using the monoclonal antibodies and methods disclosed inVetvicka et al., Biochem. Mol. Biol. Int'l. 30: 921-928, 1993,incorporated herein by reference in its entirety). In another example,one or more human mammary epithelial antigens (HME-Ags) corresponding toglycoprotein components of the human milk fat globulin (HMFG) proteinare detected in the primary mammary fluid sample, or in the biologicalsample that is used in the breast cancer marker assay, using specificantibody probes, as described by Rosner et al., Cancer Invest. 13:573-582, 1995; Ceriani et al. Proc. Natl. Acad. Sci. USA 74: 582-586,1982; Ceriani et al., Breast Cancer Res. Treat. 15; 161-174, 1990, eachincorporated herein by reference in its entirety). In many cases, thesample verification assay can be incorporated within the breast cancermarker assay in a single procedure, for example as described below inExample 4, an assay for HME-Ags (wherein the HME-Ag findings areindicative of sample origin/quality, and also of the presence and/orquantity of a specific breast cancer marker in the sample). In anotherexample, sample verification is achieved through a combinatorial (i.e.multiple marker) immunoassay targeting various cytokeratins, which canbe detected as a panel of cytokeratins specifically expressed in mammarytissue sample. (See, Nagle, J. Histochem. Cytochem. 34: 869-881, 1986,incorporated herein by reference in its entirety). One or more of thesecytokeratins (eg. K5, K8, K18 and K19) can be simultaneously orindependently measured in the context of a breast cancer assay, and thelevel of expression of the subject cytokeratin(s) can yield informationconcerning the presence or status of breast cancer in a patient. (Seefor example, Focus, Harvard University News Office, Mar. 21, 1991, pp.2-3; and Lee, Proc. Natl. Acad. Sci. 88: 1991, each incorporated hereinby reference in its entirety).

EXAMPLE 3

Cytology in Biological Samples From Mammary Fluid

This example describes the use of conventional cytological techniques toidentify and classify breast diseases from samples obtained as describedin Example 1.

Following collection of the sample in the sample collector, the centralregion of a clean glass microscopic slide is touched to the sample and acover slip is slid over the sample to spread it along the surface of theslide. The slide is allowed to air dry and then is fixed, for example inabsolute alcohol, and stained with standard cytological stains, such asmethylene blue, hematoxyln and eosin, and other suitable stains.

The slides are then examined by light microscopy for evidence ofatypical growth of cells and clumps of cells, using well known methods,including those described in Diagnosis of Non-Palpable Breast Lesions:Ultrasonographically Controlled Fine-Needle Aspiration: Diagnostic andprognostic Implications of Cytology by Jacqueline Mouriquand, publishedby S Karger Pub: July 1993, ISBN: 3805557477; Breast: Guides to ClinicalAspiration Biopsy by Tilde S. Kline, Irwin K. Kline, published byIgaku-Shoin Medical Pub: May 198g, LSBN: 0896401596; Cytopathology ofthe Breast (Asop Theory and Practice of Cytopathology; 5 by ShahlaMasood, published by American Society of Clinical Pathology: November199S, ISBN: 0891893806; Fine Needle Aspiration Cytology and Its ClinicalApplications: Breast and Lung by Philip S. Feldman, published byAmerican Society of Clinical Pathology: November 1984, ISBN: 0891891846,each incorporated herein by reference in its entirety.

EXAMPLE 4

Immunoassay for Human Mammary Epithelial Antigens in Biological SamplesFrom Mammary Fluid

Human mammary epithelial antigens (HME-Ags) are glycoprotein componentsof the human milkfat globule (HMFG) and of the membrane of the breastepithelial cell, which are released by breast tumors and not by normalbreast tissue. (Ceriani et al., Proc. Natl. Acad. Sci. 74: 582-586,1977, incorporated herein by reference in its entirety). In the presentexample, several HME-Ags, having molecular weights of 150, 70, and 45kilodaltons, are detected and measured using specific anti-HMFG oranti-human mammary epithelial (α-HME) probes prepared and employed asdescribed by Ceriani et al., Proc. Natl. Acad. Sci. 79: 5420-5425, 1982(incorporated herein by reference in its entirety).

To begin the assay, biological samples from mammary fluid collected onnitrocellulose filters are eluted electrophoretically into phosphatebuffered saline to provide a test sample, according to standard methods.Alternatively, whole mammary fluid or other types of biological samplesobtained from mammary fluid can be constituted in an appropriate mediumor mixture to provide a test sample for the assay. Once the test sampleis thus provided, it is then assayed according to the HME-Agsradioimmunoassay (RIA) methods described in Ceriani et al., BreastCancer Res. Treat. 15: 161-174, 1990 (incorporated herein by referencein its entirety).

Briefly, the RIA includes two preliminary treatments of the biologicalsamples to separate interfering factors: a centrifugation step toseparate out any fat present, and a second, precipitation step toprecipitate potential immunocomplexes using polyethyleneglycol (PEG).The next steps comprise the assay proper, where HMFG antigen bound to asolid support (microtiter plates) is presented to stoichiometric orlesser amount of the α-HME antibody probe, and binding of the α-HME iscompeted by the biological samples from mammary fluid preliminarilytreated as above. The amount of α-HME bound to HMFG antigen on the solidphase is determined in a final step by detection of the α-HME antibodyprobe by radioiodinated, affinity-purified rabbit anti-mouseimmunoglobulin.

Solutions used in the assay are as follows: i) Phosphate buffered saline(PBS): 176 ml 0.05M KH₂PO₄, 608 ml 0.05M Na₂HPO₄, and 8 g NaCl broughtup to 1000 ml in H₂O (pH7.4). ii) RIA buffer: 0.1% BSA, in 0.3%Triton-X-100 (Research Prod. International Corp., Mount Prospect, Ill.)plus 0.05% sodium azide in PBS. iii) Detergent buffer: 0.3% Triton-X-100plus 0.05% sodium azide in PBS. iv) Buffered polyethylene glycol (PEG):6.6% PEG (M.W. 8000) (Sigma) plus 0.05% sodium azide in PBS)¹²⁵I-labeled affinity-purified rabbit anti-mouse immunoglobulin(Rα-mouse Ig) (Antibodies, Inc., Davis, Calif.), radioiodinated by thechloramine-T procedure as reported (Ceriani et al., Proc. Natl. Acad.Sci. 79: 5420-5425, 1982) and diluted to 4×10⁶ cpm/ml, in RIA buffer.Rabbit polyclonal anti-HMFG antibodies or rabbit anti-human mammaryepithelial antibodies (α-HME) were prepared and assayed as described(Id.).

To prepare a standard curve for evaluating assay results, controlsamples from normal human mammary fluid (exposed to nitrocellulosefilters and eluted in the same manner as the nitrocellulose adsorbed,eluted test sample, or alternatively provided as normal whole mammaryfluid or other selected type of sample obtained from normal mammaryfluid, constituted in an appropriate medium or mixture to provide asuitable control assay sample) are centrifuged for 7 min at 10,240 rpmat 10° C. The upper white band formed at the top of the sample (if thereis one) is discarded. Fresh 100 pg protein/ml solution of lyophilizeddilipidated HMFG (Ceriani et al., Proc. Natl. Acad. Sci. 74: 582-586,1977) in detergent buffer is prepared and sonicated at 10 secondintervals for a total of 4 minutes (10 sec. of sonication, followed by a10 sec. silent period) using a double step micro tip horn at 25 watts ona Sonifier Cell Disrupter 185 (Branson, Danbury, Conn.) at 4° C. HMFGsolutions at concentrations of 0, 10, 33.3, 100, 333.3, and 1000 ngprotein/ml are prepared in spun female sera, and 3 aliquots of 180 μl ofeach HMFG level in normal female sera are pipetted into 400 μlpolyethylene microcentrifuge tubes (West Coast Sci. Emeryville, Calif.).150 μl of 6.6% PEG solution is added to each microcentrifuge tube, andthe tubes are incubated overnight on a rotating shaker at roomtemperature.

Test samples are processed in a comparable manner, by centrifuging300-350 μl of the eluted nitrocellulose filtrate in solution (or,alternatively, of mammary fluid or other assay sample alternative) in a400 μl microcentrifuge tube for 5-7 min. at 10,240 rpm at 10° C. Themicrocentrifuge tubes are then cut with a razor blade below the whiteband formed by the sera (if there was one) and 180 μl of remaining serais transferred to a new microcentrifuge tube. 150 μl of a 6.6% PEGsolution is then added to each microcentrifuge tube, and the tubes areincubated overnight on a rotating shaker at room temperature.

Day Two

(1) α-HME is diluted to its appropriate concentration in detergentbuffer. The antibody solution has stoichiometric or lesser amounts ofα-HME to 6 ng HMFG protein equivalent (prot. eq.). Six ng of HMFG iscovalently bound to microtiter plates by the methylated BSA procedurepreviously described by Ceriani, In: Kennet et al., (eds) MonoclonalAntibodies and Functional Cell Lines, Plenum Press, New York, 1984, pp.398-402, incorporated herein by reference in its entirety.

(2) To process test samples and control samples on the second day,microcentrifuge tubes are centrifuged for 7 min. at 10,240 rpm at 10° C.in a SHMT rotor with a Sorvall RC5C centrifuge. In triplicate, 55 μl ofsupernatant is pipetted into empty microtiter plate wells (Dynatech,Alexandria, Va.), and any precipitate pelleted is left undisturbed. 25μl of 6.6% PEG solution is added to each well. 30 μl of α-HME diluted indetergent buffer is also added to each well, and a non-porous Scotch®tape is placed over the wells to avoid evaporation. The microtiter plateis then incubated overnight at room temperature on a rotary shaker.

Day Three

The microtiter plates are centrifuged (3000 r.p.m.) for 30 minutes atroom temperature to decant suspended perceptible matter.

50 μl of RIA buffer is added to wells of microtiter plates containing 6ng HMFG and aspirated off after 5 minutes.

The total contents of microtiter plates from 1), save for anyprecipitation induced by the PEG and already pelleted, are carefullytransferred to the wells of another set of microtiter plates containing6 ng HMFG per well (Day 2,1), above.

The microtiter plates are incubated for 3 hours with rotating agitationat room temperature.

The plates are washed 5 times with RIA buffer using Dynadrop SR-1automatic dispenser form Dynatech.

50 μl of the radioiodinated affinity-purified rabbit anti-mouseimmunoglobulin diluted in RIA buffer is then adder per well.

The plate is covered with tape and incubated with rotating agitation for2 hours at room temperature.

The plate is washed 5 times with RIA buffer.

Wells are cut and counted in a gamma counter.

The results of these assays will yield important information concerningthe presence and/or status of cancer in patients, comparable in scopeand value to the data provided by serum assays conducted for the HME-Agbreast cancer marker by Ceriani et al., Breast Cancer Res. Treat. 15:161-174, 1990. By selecting patient and control samples and developingand evaluating comparative data according to the procedures followed byCeriani and his coworkers, the assay methods of the invention will alsobe readily adapted for use in direct clinical applications to determineboth prognostic and treatment related variables in breast cancerpatients. Reagents and conditions for the assays can of course besubstituted or adjusted depending on a variety of anticipated variables,by applying well known immunological methods and principles.

EXAMPLE 5

Competitive Radioimmunoassay for Non-Penetrating Glycoprotein inBiological Samples From Mammary Fluid

This competitive radioimmunoassay is based on the displacement by breastepithelial antigens contained in biological samples from mammary fluidobtained according to the methods of the invention of the binding ofstoichiometric or lesser quantities of the monoclonal antibody Mc5 to asolid-phase-bound antigen known as non-penetrating glycoprotein (NPGP)contained in HMFG. HMFG is bound to a solid support and exposed to theMc5 antibody during an incubation period allowing the antibody to bindthe NPGP antigen in solid phase-bound HMFG. The presence and/or level ofNPGP in the biological sample is ultimately examined by ability of thesample to compete for Mc5 binding to the NPGP antigen in the solidphase-bound HMFG, as detected and/or measured using a radiolabeled goatanti-mouse antibody to bind and label the Mc5 antibody probe.

Buffer and other solutions and reagents in this example are generallythe same as those used for the HME-Ags polyclonal antibodyradioimmunoassay described in Example 4, above. To provide test samplesfor the assay, biological samples from mammary fluid contained onnitrocellulose filters are eluted electrophoretically into phosphatebuffered saline, according to standard methods. Alternatively, wholemammary fluid or other types of biological samples obtained from mammaryfluid can be constituted in an appropriate medium or mixture to providea test sample for the assay. Once the test sample is thus provided, itis then assayed according to the NPGP/Mc5 radioimmunoassay (RIA) methodsdescribed in Ceriani et al., Breast Cancer Res. Treat. 15: 161-174, 1990(incorporated herein by reference in its entirety), as follows:

400 μl of pooled normal female mammary fluid (exposed to nitrocellulosefilters and eluted in the same manner as the nitrocellulose adsorbed,eluted test sample, or alternatively provided as normal whole mammaryfluid or other types of biological samples obtained from normal mammaryfluid constituted in an appropriate medium or mixture to provide a testsample) to provide a suitable control sample, which is diluted to 2.4 mlusing RIA buffer at a 1:6 concentration.

A 500 μg/ml solution of lypholized HMFG is prepared in 1×PBS with 0.3%Triton-X-100, 0.05% sodium azide, and sonicated using a double stepmicro tip horn at 25 watts on a Sonifier Cell Disrupter 185 (Branson,Danbury, Conn.) for 4 minutes (10 sec. sonication, 10 sec. silentperiod, at 4° C.).

Solutions to prepare a standard curve are prepared using the 2.4 ml 1:6normal female serum and increasing amounts of HMFG (0, 0.25, 2.5, 25, 50pg/ml HMFG, as described above in Example 4).

Each test assay sample is diluted 1:6 with RIA buffer (40 μl of serum to200 μl RIA buffer) to form a diluted test assay sample, and vortexed.

Mc5 stock solution is prepared so that it contains less thanstoichiometric amounts of antibody to 100 ng protein/well of HMFGcovalently bound to microtiter plates prepared as previously describedby Ceriani, In: Kennet et al., (eds) Monoclonal Antibodies andFunctional Cell Lines, Plenum Press, New York, 1984, pp. 398-402,incorporated herein by reference in its entirety

200 μl RIA buffer are added to each well of 100 ng HMFG and thenaspirated after 5 minutes.

To prepare a standard curve, 30 μl of HMFG standardizing solutions (asin 3 above) are added in quadruplicate to a 100 ng protein/well HMFGmicrotiter well.

30 μl of diluted test assay sample (or, alternatively, of mammary fluidor other assay sample alternative) are added in triplicate to 100ng/well HMFG microtiter wells.

To each well 20 μl of the Mc5 stock solution is added.

Microtiter plates are covered with nonporous Scotch® tape and incubatedovernight at room temperature on a rotating agitator.

The next day the wells are aspirated and washed 5 times with RIA buffer.

To each well 50 μl of 200,000 cpm/50 μl ¹²⁵I-goat anti-mouse antibodyare dispensed. The wells are covered with nonporous tape and placed on arotating agitator for 3 hours at room temperature.

Wells are washed 5 times with RIA buffer.

Each well is cut and the radioactivity is counted using a gamma counter.

The results of these assays will yield important information concerningthe presence and/or status of cancer in patients, comparable in scopeand value to the data provided by serum assays conducted for the NPGPbreast cancer marker by Ceriani et al., Breast Cancer Res. Treat. 15:161-174, 1990. By selecting patient and control samples and developingand evaluating data according to the well known procedures followed byCeriani and his coworkers, the assay methods of the invention will bereadily adapted for use in direct clinical applications to determineboth prognostic and treatment related variables in breast cancerpatients. As will be understood by those skilled in the art, reagentsand conditions for the assays can be substituted or adjusted dependingon a variety of anticipated variables, according to well knownimmunological methods and principles.

EXAMPLE 6

Solid Phase Immunoassay for Mucinous Carcinoma Associated Antigen inMammary Fluid

This example uses a sensitive, solid phase immunoassay to detect themucinous carcinoma associated antigen (MCA) in biological samples frommammary fluid obtained according to the methods of the invention. MCAconcentrations are determined using an antibody-bead immunoassay kitprovided by Hoffman-La Roche (Basel, Switzerland), and using thereagents and procedures provided by the manufacturer and described infurther detail in Eskelinen et al., Anticancer Res. 9: 437-440, 1989.Briefly, test assay samples of whole mammary fluid and standards arefirst incubated with MCA monoclonal antibody beads and then, afterappropriate washings, enzyme (horseradish peroxidase) labeled secondaryantibody is added. During the second incubation the anti-MCA enzymeconjugates are attached to the antibody antigen complex on the beads.

Excess conjugates are removed by washing and, finally, enzyme substrateare added and the color formed is recorded. The solid phase assay formatpresented in this example can be adapted for use in a wide array ofother assays to detect and/or measure other cancer markers besides theMCA marker, with enhanced sensitivity. In addition, the results of theseassays can be evaluated along with those of complementary assaysdetecting and/or measuring different markers to yield more preciseinformation concerning the presence and/or status of cancer in patients,as exemplified by the combinatorial MCA/CA 15-3 assays described byEskelinen et al., Anticancer Res. 9: 437-440, 1989; see also Eskelinenet al., Anticancer Res. 8: 665-668, 1988, each incorporated herein byreference in its entirety.

EXAMPLE 7

Western Analysis of Proteins From Cellular Fractions of Human MammaryFluid Using Polyclonal and Monoclonal Antibody Probes to DetectVasopressin

A variety of assays are provided by the present invention that focus oncellular samples from human mammary fluid. In general, these assays relyon isolation by standard separation methods (eg. centrifugation, sucrosegradient, etc.) of cells, membranes or other cell components from wholemammary fluid expressed according to the above methods. Biologicalsamples containing whole cells from expressed mammary fluid areparticularly useful for cytological and cytochemical examination todetect and evaluate breast cancer in patients. Biological samplescontaining purified cell membrane fractions from human mammary fluid areparticularly useful in this context, for example to detect and/ormeasure breast cancer markers that are expressed by alveolar-ductalcells as intracellular or membrane bound proteins and are therefore notas readily detected in liquid fractions of mammary fluid as secretedproteins.

The present example focuses on assays for detecting the peptide hormonevasopressin in biological samples from mammary fluid, using methodsadapted from North et al., Breast Cancer Res. Treat. 34: 229-235, 1995.Specifically, this assay uses a test sample of crude protein isolatedfrom a pooled sample of cells obtained from expressed mammary fluid. Thecells are separated from whole mammary fluid according to standardmethods, and crude protein is extracted from the cells by sonication in100 volumes of 0.1 M HCl. The resulting protein suspensions are thencentrifuged at 1500×g for 10 min. at ambient temperature, and solubleprotein is precipitated with 40% TCA. This protein is pelleted bycentrifugation at 10,000×g for 2 min. TCA is then removed from pelletsby washing (×2) with ether. Protein is resuspended in 0.1 M Tris HCl(pH8.7), reduced with mercaptoethanol at 100° C. for 5 min. (and in somecases S-alkylated with N-ethyl maleimide), and subjected to SDS-PAGEelectrophoresis on 15% gels at pH 9.3 using the method of Laemeli,Nature 227: 680-685, 1970, incorporated herein by reference in itsentirety. Separated proteins are then electrophoretically transferredwith 20 mM Tris glycine (pH 8.0) to Immobilon PVDF membranes (Millipore,Bedford, Mass.). These membranes are blocked with a 5% non-fat milksolution, washed (1×15 min., 2×5 min.) with PBS containing 0.5% Triton,and incubated with preparations of mouse monoclonal antibody to VP-HNP,with rabbit polyclonal antibodies to VP, with rabbit polyclonalantibodies to VAG, or with ubiquitous mouse or rabbit IgG (negativecontrols) (for description of antibodies and antibody preparation seeNorth et al., Breast Cancer Res. Treat. 34: 229-235, 1995, incorporatedherein by reference in its entirety), for 1 h at ambient temperature.Following a second wash in PBS-Triton (1×15 min., 2×5 min.), themembranes are treated, respectively, with goat anti-mouseIgG-horseradish peroxidase conjugate or goat anti-rabbit IgG-horseradishperoxidase conjugate for 1 h, and then washed with PBS-Triton (1×15min., 4×5 min.). Immunoreactive proteins are visualized using an ECLWestern Blotting Detection System with exposure of x-ray film from 10seconds to 5 min. Prestained SDS-PAGE standard proteins are employed asmolecular size markers.

Recent studies suggest that vasopressin is universally expressed inbreast carcinoma and is absent from normal breast cells. North et al.,Breast Cancer Res. Treat. 34: 229-235, 1995. These and other resultsindicate that vasopressin and its relatives are important breast cancermarkers which can be readily detected using immunological assays ofproteins isolated from breast tumor cells. Accordingly, the results ofthe present example using cell samples isolated from human mammary fluidare also expected to yield important information concerning the presenceand/or status of cancer in patients.

EXAMPLE 8

Quantification of Carcinoembryonic Antigen in Biological Samples FromMammary Fluid by Dot Immunoblotting Assay

Among the more sensitive assays of the invention, useful 25 formeasuring low levels of breast cancer markers and for detecting markerswhen only small volumes of expressed mammary fluid are available, is thedot immunoblotting assay. In the present example, carcinoembryonicantigen (CEA) is measured in whole mammary fluid using an ELMOTECH®anti-CEA monoclonal antibody kit (Mochid Pharmaceutical Co., Tokyo,Japan) in a dot blot assay format. Briefly, anti-CEA monoclonal antibodyis diluted to appropriate concentrations and coated on the plastic film.Aliquots (5 μl) of either standard CEA solution (0, 100, 200, and 500ng/ml), or of the whole mammary fluid assay sample, are smeared on theimmobilized film. Assay standards are prepared from purified antigenpreparations, in accordance with the ELMOTECH® kit manufacturer'sinstructions. If necessary, 1000 ng/ml CEA solution is also used as astandard. After drying at room temperature, the film is exposed toperoxidase-conjugated anti-CEA antibody for 20 min at room temperature.The film is then washed extensively with 1 M saline containing 0.5%(v/v) Tween 20. The enzyme reaction is visualized usingtetramethylbenzidine as a chromogen. The developing solution consists of0.05 mM tetramethylbenzidine and 0.01% hydrogen peroxide in McIlvainbuffer (0.1% M phosphate-citrate buffer), pH 5.0, containing 10%methanol. The concentration of CEA in the mammary fluid assay sample isdetermined by comparing the color intensities with a correspondingstandard.

The assay disclosed in the present example, and related assaysincorporating antibodies to other tumor markers besides CEA, areparticularly useful for measuring low levels of breast cancer markersand for detecting markers in limited sample volumes. The results ofthese assays will yield important information to determine bothprognostic and treatment related variables in breast cancer patients. Aswill be understood by those skilled in the art, reagents and conditionsfor the assays can be substituted or adjusted depending on a variety ofanticipated variables, according to well known immunological methods andprinciples.

EXAMPLE 9

Detection of Procathepsin D and Cathepsin D Activity in BiologicalSamples From Mammary Fluid

Cathepsin D is a lysosomal aspartic proteinase which has been studiedintensively as a marker for cancer processes necessary for metastasis.In the present example, polyclonal antibodies against procathepsin D areused to immunoprecipitate and immunochemically detect proteins fromwhole mammary fluid or cell lysates from mammary fluid, generallyaccording to the methods disclosed in Vetvicka et al., Biochem. Mol.Biol. Int'l. 30: 921-928, 1993 (incorporated herein by reference in itsentirety). Alternatively, or as a complementary assay, the proteaseactivity of cathepsin D is detected, also according to the methodsdisclosed in Vetvicka et al. (Id.). Briefly, pooled whole mammary fluid(preferably 3 ml if available) is diluted with 3 ml of buffer A (50 mMTris.HCl, 5 mM CaCl₂, 1 mM MgCl₂, 500 mM NaCl pH 7.2). The suspension iscentrifuged for 30 minutes at 10,000 g. The resulting water phase iscentrifuged again under the same conditions. The soluble part (total ofapproximately 4.5 ml) is loaded on a 1 ml column of Concanavalin ASepharose (Pharmacia, Uppsala, Sweden) equilibrated in buffer A, andafter washing with buffer A the retained proteins are eluted using 0.75M methyl α-D-mannopyranoside. The fractions (250 μl) are analyzed forcathepsin D activity using the ¹⁴C hemoglobin assay as described by Linet al., J. Biol. Chem. 264: 4482-4489, 1989 (incorporated herein byreference in its entirety), by western blots and by silver-stainedelectrophoresis. The inhibition of human milk procathepsin D isaccomplished by adding 2 μl of 1 mM pepstatin A (Boehringer Manheim,Germany) dissolved in methanol to the reaction mixture.

This assay provides but one example of many possible embodiments of theinvention that incorporate known biochemical assays, in addition to, orsupplemental to immunological assays, to evaluate biological samplesfrom mammary fluid to determine cancer related variables. Thefundamental methods provided herein for obtaining samples from humanmammary fluid render these assays readily adaptable for widespreadclinical application to detect and/or measure the activity of a subjectbreast cancer marker within a non-invasive screening protocol.

Those with ordinary skill in the art will appreciate that otherembodiments and variations of the invention are possible which employthe same inventive concepts described above. Most particularly, a wideand rapidly expanding array of useful breast cancer markers (includingproteins, DNA and RNA sequences and other markers) and probes (includingimmunological, nucleotide and biochemical probes) are readily availablefor adaptation and use within the methods and kits of the invention.These markers and probes are described or referenced to a large extentin the literature cited and incorporated within the present disclosure,or are elsewhere published in the literature or well known in the art.Among these known and emerging markers and probes, useful exampleswithin the invention include Her 2 (also known as erbB-2 and neu). Her 2is a transmembrane glycoprotein growth factor receptor of the EGFreceptor family encoded by a gene located on chromosome 17q, a region offrequent amplification in breast cancer cell lines. This marker ishighly predictive of breast cancer and can be detected in cellularsamples of the invention using known nucleotide probes to detect geneticdefects in Her 2, or to detect and/or measure mRNA to determineoverexpression of Her 2 linked to increased proliferation of cancercells. (See for example, Visscher et al., In Weinstein and Graham (eds)Advances in Pathology and Laboratory Medicine, vol 5, St Louis, MosbyYuear Book, 1992, pp. 123-161; Barbareschi et al., Am. J. Clin. Pathol.98: 408-418, 1992; Slamon et al., Science 235: 177-182, 1987; eachincorporated herein by reference in its entirety). Protein levels of Her2 are also readily detected using available immunological probes. (Forreview see Porter-Jordan et al., Hematol. Oncol. Clin. North Amer. 8:73-100, 1994 and articles cited on page 80 therein, each incorporatedherein by reference in its entirety). Additional markers for use withinthe invention include EGF and the EGF receptor, for which immunologicaland non-immunological probes and assay methods readily adaptable withinthe invention are characterized in detail at page 80-81 of Porter-Jordanet al., Hematol. Oncol. Clin. North Amer. 8: 73-100, 1994 and in thereferences cited therein, each incorporated herein by reference in itsentirety. Additional examples of proliferation markers, growth factorsand receptors, proteases, adhesion factors, angiogenic factors,oncogenes and tumor suppressor genes that provide useful breast diseasemarkers and probes within the methods and kits of the invention includeKi67 Growth Factor, Cyclin D1, Proliferating Cell Nuclear Antigen,Transforming Growth Factor, Tissue Plasminogen Activator, Insulin GrowthFactor Receptors, Collagenase Type IV, Laminin Receptor, Integrins, p53,rb, nm23, ras, c-myc, c-myb, Heat Shock Proteins, Prolactin,Neuron-Specific Enolase, IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actin,IL-10, S-100 protein, Vimentin, Epithelial Membrane Antigen, bcl-2,CAl5-3, CA 19-9, Tn Antigen, Alpha-lactalbumin, LASA, Gal-GalNAC,GCDFP-15, Le(y)-Related Carbohydrate Antigen, CA 125, uPA, uPA relatedantigens and complexes, uPA Receptor, PAl-1 and PAl-2,Betaglucuronidase, CD31, CD44 splice variants, blood group antigensincluding ABH, Lewis, and MN, and genetic lesions or altered expressionlevels of CCND1, EMS1, BRCA1 and BRCA2 genes, and many others, for whichimmunological and non-immunological binding partners, probes and assaymethods are known and readily adaptable within the invention. Inaccordance with the foregoing disclosure, the invention is not to belimited by the exemplary description and drawings herein, but is to bedetermined in scope by the claims which follow.

We claim:
 1. A sample collection device for collecting a biologicalsample from a mammary organ of a patient, comprising: a breast engagingmember constructed of a non-porous material sized and dimensioned toreceive at least a nipple portion of a breast of said patient and form asuction seal therewith: a solid phase sample collection medium in fluidconnection with said breast engaging member for receiving a sample ofexpressed breast fluid; and vacuum pump means in gaseous connection withsaid breast engaging member for generating negative pressure through thebreast engaging member to facilitate breast fluid expression, whereinsaid solid phase sample collection medium is selected from the groupconsisting of microscopic glass slides, capillary tubes, collectiontubes, columns, micro-columns, wells, plates, membranes, filters,resins, inorganic matrices, beads, particulate chromatographic media,plastic microparticles, latex particles, coated tubes, coated templates,coated beads, coated matrices, or a combination thereof.
 2. The samplecollection device of claim 1, including removable coupling means forremovably coupling said sample collection housing with said breastengaging member.
 3. The sample collection device of claim 1, whereinsaid solid phase sample collection medium is supported by a supportmember integrally or removably mounted within said sample collectionhousing in fluid connection with said breast engaging member.
 4. Thesample collection device of claim 3, wherein said support member isdisc-shaped and is interposed between said breast engaging member andsaid sample collection housing.
 5. The sample collection device of claim3, wherein said support member has upper and lower retaining rings andsupports a sheet of absorbent or adsorbent material.
 6. The samplecollection device of claim 3, wherein said support member supports asolid phase sample collection template selected from capillary tubes,coated tubes, columns, micro-columns, plates, wells and microscopicslides, or a combination thereof.
 7. The sample collection device ofclaim 3, wherein said support member defines a fluid-retaining well. 8.The sample collection device of claim 3, wherein said support memberincludes at least one air channel to allow negative pressure to passthrough the air channel to and from said breast engaging member.
 9. Thesample collection device of claim 1, wherein said solid phase samplecollection medium is a particulate medium contained within a cartridgeremovably mounted within said sample collection housing and having afirst end of said cartridge in fluid connection with said breastengaging member.
 10. The sample collection device of claim 9, whereinsaid first end of said cartridge is covered by a porous barriermaterial.
 11. A sample collection device for collecting a biologicalsample from a mammary organ of a patient, comprising: a breast engagingmember constructed of a non-porous material sized and dimensioned toreceive at least a nipple portion of a breast of said patient and form asuction seal therewith; a solid phase sample collection medium in fluidconnection with said breast engaging member for receiving a sample ofexpressed breast fluid; reciprocating means for reciprocally adjusting aposition of said solid phase sample collection medium relative to saidbreast engaging member; and vacuum pump means in gaseous connection withsaid breast engaging member for generating negative pressure throughsaid breast engaging member to facilitate breast fluid expression. 12.The sample collection device of claim 11, wherein said reciprocatingmeans incorporates a support member or carrier reciprocatingly mountedrelative to said breast engaging member, said support member or carriersupporting the solid phase sample collection medium.
 13. The samplecollection device of claim 11, wherein said solid phase samplecollection medium is supported by a support member removably mounted influid connection with said breast engaging member.
 14. The samplecollection device of claim 13, wherein said support member is removablymounted to a carrier reciprocatingly mounted within a sample collectionhousing fluidly connected with said breast engaging member.
 15. Thesample collection device of claim 11, wherein said reciprocating meansinclude a rotating member of a sample collection housing sealably androtatably interconnected with said breast engaging member, said rotatingmember having a lumenal, helically oriented groove dimensioned toreceive a riding peg extending transversely from a support member orcarrier supporting said solid phase sample collection medium and alongitudinally oriented, lumenal groove dimensioned to receive anangularly fixating keel extending transversely from said carrier orsupport member, whereby rotation of the rotating member of the housingdrives rotation of the carrier or support member and translates theriding peg along the helical groove to cause the support member orcarrier to reciprocate forward or backward relative to the breastengaging member.
 16. The sample collection device of claim 15, whereinsaid rotating member of said housing is removably interconnected withsaid breast engaging member to facilitate insertion and removal of saidsolid phase sample collection medium.
 17. The sample collection deviceof claim 11, wherein said reciprocating means include a slide mechanism.18. The sample collection device of claim 17, wherein said slidemechanism includes a manifold defining an inner lumen closed at one endto prevent gaseous connection with an outer lumen of a sample collectionhousing through which vacuum pressure is transmitted from said pumpmeans to said breast engaging portion, and a sliding support member orcarrier supporting said solid phase sample collection medium slideablymounted within said inner lumen.